ALBERT

Description: Introduction: The currently used unfractionated heparin (UFH) and low molecular weight heparins (LMWH) are mostly derived from Porcine mucosal tissue. Since the technology to manufacture heparin has advanced and the quality assurance practices are in place, improved products with high potency and purity are now available. Considering these factors the resourcing of heparins utilizing bovine (cow) and ovine (sheep) tissues is discussed at regulatory and pharmaceutical levels. The purpose of this study is to compare multiple individual batches of UFH obtained from Bovine, Ovine, and Porcine origin and their depolymerized product obtained by benzylation followed by alkaline hydrolysis representing enoxaparins. Materials and Methods: The molecular profile of the heparins and enoxaparins from various sources were determined using the size exclusion chromographic method. The anticoagulant potency was measured using clot based methods such as aPTT and Thrombin Time. The AT mediated anti-Xa and anti-IIa activities were also measured in defined biochemical sysytems. A comercially obtained chromogenic substrate based methods were used to determine the USP potency in terms of anti-Xa and anti-IIa activities (Hyphen Biomedical, Ohio, USA). The relative interaction of the heparins and enoxaparins with heparin induced thrombocytopenia (HIT) antibody induced aggregation of platelets were investigated using serum pool obtained from clinically confirmed HIT cases using aggregometry. Results: The molecular profile of multiple batches of the Bovine, ovine, and porcine heparins and enoxaparin were almost comparable and ranged from 15-18 kDa. The global anticoagulant and amidolytic protease assays for the bovine heparin were consistently lower than porcine and ovine samples. In the purified system the Porcine and Ovine preparations consistently showed lower IC50 values for both the thrombin and Xa inhibition in contrast to the bovine heparin. Similar trends were observed in the anti IIa assays. The USP potency of 28 batches of porcine ranged from 170-210 U/mg, whereas the 21 batches of ovine heparins exhibited comparable potencies in the range of 160-210 U/mL. In contrast the USP potency of 30 batches of bovine mucosal heparin was much lower and ranged from 110-140 U/mg. The anti-Xa - IIa ratio for the porcine and ovine heparins were comparable. However, the anti-Xa - IIa ratio of bovine heparin was somewhat lower. The ovine and porcine enoxaparin exhibited comparable potencies which ranged from 94-110 U/mg whereas bovine enoxaparin was slightly lower, ranging from 80-87 U/mg. However the antiXa and anti-IIa ratios of the enoxaparins derived from various species were comparable. In the HIT screening, there was no difference between the HIT responses in the heparins from different species. Similar results were obtained amongst enoxaparins of different origins. Conclusions: These studies show that heparins from bovine, ovine, and porcine origin exhibit comparable molecular profiles. While the porcine and ovine heparins exhibit similar biological potencies, the bovine heparin was found to be weaker. The enoxaparins derived from these species also exhibit similar molecular profiles, however, in functional assays, they exhibited similar trends where the bovine derived products were weaker. In the HIT screening profile, the heparins from all of these species produced stronger effects in comparison to their enoxaparin counterparts. These results suggest that heparin and enoxaparin derived from ovine mucosal tissue exhibit comparable biosimilar profiles. However, the bovine heparins and enoxaparin derived from bovine mucosal tissues are somewhat weaker. Disclosures Yao: Ronnsi Pharma: Employment.

Description: Background: Most research on prognosis after PE has focused on outcomes such as mortality and PE recurrence, while patient-centered outcomes such as quality of life (QOL), dyspnea and walking capacity have been largely unstudied. To address this knowledge gap, we performed the ELOPE (Evaluation of Longterm Outcomes after PE) study, a prospective, observational, multicenter cohort study of long-term outcomes after acute PE (www.clinicaltrials.gov NCT01174628). Objectives: In this extended analysis of ELOPE data, we assessed clinically meaningful improvement and predictors of change in QOL, dyspnea and walking capacity during the year following PE diagnosis. Methods: Patients aged 18 years or older with a 1st episode of acute PE diagnosed within the previous 10 days screened at 5 Canadian recruiting centers were potentially eligible to participate. Exclusion criteria were subsegmental-only PE, preexisting severe cardiopulmonary comorbidity, previous proximal DVT, contraindication to CT pulmonary angiography, life expectancy

Description: Key Points The secreted lymphangiogenic protein CCBE1 is essential for fetal but not postnatal erythropoiesis. Loss of CCBE1 impairs erythroblastic island formation and function.

Description: Thrombosis can be initiated when activated platelets adhere to injured blood vessels via the interaction of subendothelial collagen with its platelet receptor, glycoprotein (GP) VI. Here we observed that incubation of platelets with convulxin, collagen, or collagen-related peptide (CRP) resulted in GPVI signaling-dependent loss of surface GPVI and the appearance of an approximately 55-kDa soluble fragment of GPVI as revealed by immunoblotting. Ethylenediaminetetraacetic acid (EDTA) or GM6001 (a metalloproteinase inhibitor with broad specificity) prevented this loss. In other receptor systems, calmodulin binding to membrane-proximal cytoplasmic sequences regulates metalloproteinase-mediated ectodomain shedding. In this regard, we have previously shown that calmodulin binds to a positively charged, membrane-proximal sequence within the cytoplasmic tail of GPVI. Incubation of platelets with calmodulin inhibitor W7 (150 μM) resulted in a time-dependent loss of GPVI from the platelet surface. Both EDTA and GM6001 prevented this loss. Surface plasmon resonance demonstrated that W7 specifically blocked the association of calmodulin with an immobilized synthetic peptide corresponding to the calmodulin-binding sequence of GPVI. These findings suggest that disruption of calmodulin binding to receptor cytoplasmic tails by agonist binding to the receptor triggers metalloproteinase-mediated loss of GPVI from the platelet surface. This process may represent a potential mechanism to regulate GPVI-dependent platelet adhesion.

Description: A murine monoclonal antibody specific for glycoprotein (GP)IIIa was prepared by immunization with a GPIIb- and GPIIIa-enriched Triton X-114 extract of platelet membranes. This antibody, designated AP-3, was shown by indirect immunoprecipitation to react solely with GPIIIa derived from either P1A1-positive or -negative individuals. The epitope on GPIIIa recognized by AP-3 is expressed on dissociated GPIIIa as well as on Ca+2-dependent complexes of GPIIb and GPIIIa, as shown by crossed immunoelectrophoresis in the presence or absence of EDTA. A previously described monoclonal antibody specific for the GPIIb/IIIa complex (AP- 2) inhibited platelet aggregation induced by ADP, thrombin, collagen, or arachidonic acid (Pidard et al, J Biol Chem 258:12582–12586, 1983). In contrast, AP-3 had no effect on aggregation induced by any of these reagents, a finding similar to that previously reported for the GPIIb- specific monoclonal antibody, Tab (McEver et al, J Clin Invest 66:1311- 1318, 1980). At saturation, 40,200 AP-3 molecules were bound per platelet, a value similar to that obtained for AP-2 or Tab. Thus, data derived using AP-3 indicate that significant amounts of free GPIIIa are not present, thereby supporting the hypothesis that GPIIb and GPIIIa exist complexed in a 1:1 stoichiometry in the plasma membrane of intact, nonactivated platelets.

Description: Key Points Truncating PPM1D mutations confer chemotherapy resistance, leading to the selective expansion of PPM1D-mutant cells in vitro and in vivo. PPM1D inhibitor treatment reverses the chemotherapy-resistance phenotype and selectively kills PPM1D-mutant cells.

Description: Introduction: The postthrombotic syndrome (PTS) is the most common chronic complication of deep venous thrombosis (DVT). Inflammation markers can predict PTS when measured at discrete time points after DVT; however, this approach does not recognize patterns of change over time. Objective: To describe change of inflammation marker levels after acute proximal DVT and assess the association of individual biomarker trajectories with risk of PTS. Methods: The BioSOX is a substudy of the SOX Trial. Patients with first, symptomatic, proximal DVT were followed for 24 months. The study end point, PTS, was diagnosed starting from 6 months post DVT using the Villalta scale. During the SOX trial we collected blood samples from participants at baseline, 1 and 6 months, and measured concentrations of C-reactive protein (CRP), interleukin (IL)-6, IL-10 and intercellular adhesion molecule (ICAM)-1 using validated established methods. We used group-based trajectory modeling (GBTM) to identify biomarker level trajectories from the time of acute DVT up to six months post DVT and assign patients to trajectory groups. Chi-square and ANOVA tests were used to investigate the association of clinical and demographic characteristics and trajectory group assignment. Modified Poisson regression models were used to estimate risk ratios (RR) for PTS according to trajectory group. Models were adjusted for predefined covariates (age, sex, body mass index (BMI) and extent of DVT) and data driven confounders found to be significantly associated with trajectory group. Results: Of 703 patients, 327 developed PTS. Figure 1 depicts the best trajectory models for each of the 4 biomarkers. For both ICAM-1 and IL-10, the best model was a 3-group model with high, intermediate and low biomarker level trajectories. For both IL-6 and CRP, the best model was comprised of 4 groups, which consisted of the highest group (group 3) having a rapidly declining profile to an intermediate level by the first month after DVT, and a second high group (group 4) having levels that remained high for the entire sampling time. Clinical and demographic variables most closely associated with trajectory group assignment were age, BMI, infectious or inflammatory conditions in the month prior to DVT, smoking, cancer related DVT and anatomical extent of DVT. For ICAM-1 there was a significant association of trajectory group assignment and risk of PTS (Table 1). Adjusted RR for trajectory group 2 vs. group 3 was 0.79 (95% confidence interval 0.67 – 0.93). There were no associations of the other biomarker trajectory groups with PTS risk. Conclusion: To our knowledge, this is the first study to use trajectory analyses to describe temporal patterns of change in inflammation marker levels after acute DVT and relate these to risk of PTS. Results suggest that patients demonstrate distinct patterns of change in biomarker levels over the first six months after acute DVT. Persistently high ICAM-1 levels in the first six months after DVT were associated with an increased risk of PTS. Verification of these results is needed by other studies. Figure 1. Group based trajectory models for biomarker trajectories after acute DVT. Figure 1. Group based trajectory models for biomarker trajectories after acute DVT. Table 1: Association between biomarker trajectory group and PTS Trajectory group* Crude RR (95% CI) Adjusted† RR (95%CI) ICAM-1 1 vs. 3 0.60 (0.33-1.10) 0.67 (0.36-1.24) 2 vs. 3 0.74 (0.63-0.87) 0.79 (0.67-0.93) IL-10 1 vs. 3 1.01 (0.60-2.02) 1.19 (0.66-2.16) 2 vs. 3 1.08 (0.79-1.46) 1.13 (0.83-1.53) CRP 1 vs. 4 0.80 (0.60-1.04) 0.95 (0.70-1.28) 2 vs. 4 0.91 (0.73-1.13) 1.00 (0.80-1.25) 3 vs. 4 0.79 (0.61-1.01) 0.89 (0.68-1.16) IL-6 1 vs. 4 0.84 (0.62-1.15) 1.01 (0.73-1.40) 2 vs. 4 1.01 (0.76-1.35) 1.02 (0.76-1.36) 3 vs. 4 0.90 (0.62-1.32) 0.96 (0.65-1.41) ICAM-1, intercellular adhesion molecule1; IL-10, Interleukin 10; CRP, C-reactiveprotein; IL-6, Interleukin 6; RR, risk ratio. *See Figure 1 for graphical depiction of trajectory group patterns over time †Adjustment variables: ICAM-1: age, sex, BMI, extent of DVT, smoking status and type of DVT IL-6 and IL-10: age, sex, BMI, extent of DVT, infection or inflammatory condition in the month prior to DVT, smoking status and type of DVT CRP: age, sex, BMI, extent of DVT, infection or inflammatory condition in the month prior to DVT and type of DVT. Disclosures No relevant conflicts of interest to declare.

Description: Introduction The post thrombotic syndrome (PTS) is a chronic condition that develops in 20–40% of deep vein thrombosis (DVT) patients. While risk factors that predispose to the development of venous thromboembolism are widely known, factors that influence the development of PTS after DVT have not been well elucidated. Identification of factors to facilitate individualized risk assessment for PTS and thereby the need for prophylactic intervention is desirable. Objectives We conducted a systematic review to determine whether biomarkers of fibrinolysis and endothelial dysfunction can predict the risk of developing PTS among patients diagnosed with DVT. Methods Studies were identified by searching the electronic databases PubMed, EMBASE, Scopus and Web of science. Studies published between January 1990 and January 2013 which measured biomarker levels in blood of adult patients with DVT, and reported rates of PTS development in these patients were included. Extracted data included source and baseline characteristics of study subjects, potential confounders that could influence the result of measured marker (e.g. malignancy, use of anticoagulants), type of biomarker, time point(s) and method of measurement, criteria for PTS diagnosis, and measure of association including adjustment variables. Risk of bias was assessed using Newcastle-Ottawa Scale, with some additional quality items specific to this literature added. Results Out of 2376 records included in our primary screen of titles and abstracts, 84 articles were assessed for eligibility. Fourteen studies were included in this systematic review: 11 investigated the association between D-Dimer and PTS, three examined fibrinogen level, two measured Von Willebrand factor (VWF) antigen and activity, one study measured plasminogen activator inhibitor (PAI)-1, one assessed A Disintegrin and Metalloprotease with Thrombospondin type 1 repeats (ADAMTS)-13 antigen and activity and one study measured Factor XIII (FXIII) activity. Studies varied with regards to inclusion and exclusion criteria (e.g. first DVT, malignancy, current anticoagulant use), use of validated scales to diagnose PTS (e.g. Villalta scale or clinical, etiological, anatomical and pathological [CEAP] classification), timing of PTS assessment, time point of biomarker measurement after DVT, measurement method, and cut off value used for analysis of association between marker and outcome. Figure 1 summarizes results from 10 studies that reported on the association between D-Dimer and PTS. We used crude Odds Ratios (OR) as the summary measure, as adjusted ORs were available for only a few studies, and adjustment variables differed between studies. Furthermore, as some studies restricted their population on certain clinical characteristics, crude ORs may already reflect a certain degree of control for confounding. In general, adjusted ORs (where available) tended to attenuate the association. Studies were grouped according to the time point of D-Dimer measurement after DVT. Even after such subgrouping, we were still unable to combine the results within subgroups due to marked clinical heterogeneity. Three studies measured fibrinogen levels, two on the day of DVT diagnosis and one a median of 28 months after DVT. None found a significant association between fibrinogen level and the development of PTS. Similarly, no association with PTS was found in the studies examining ADAMTS-13 antigen level and activity, VWF antigen and activity, or PAI-1. The one available study that measured FXIII activity found it to be significantly lower in patients with PTS. Conclusions We systematically reviewed the literature on the association between markers of fibrinolysis and endothelial dysfunction and the development of PTS in patients after acute DVT. An elevated D-dimer level may be an intrinsic marker of residual thrombus and/or persistent activation of clotting or inflammatory pathways that could increase the propensity to develop PTS. Similarly, impaired fibrinolysis and endothelial dysfunction are pathophysiological components of PTS. Whether these markers might be useful to predict the development of PTS after DVT is still unclear. Larger prospective studies using validated scores to diagnose PTS, strict inclusion and exclusion criteria, and accounting for confounders in the design and analysis are needed. Disclosures: No relevant conflicts of interest to declare.

Description: 2980 Poster Board II-954 Background. There are few studies that examine incidence trends of venous thromboembolism (VTE) among the elderly, and moreover data on changes in the prevalence over time of VTE risk factors such as hospitalization are limited. Objectives. Using the Province of Québec's administrative health claims (“RAMQ”) and hospitalization (“MED ECHO”) databases, we determined among individuals 65 years of age and older the trend in annual VTE incidence over a ten-year period, and examined the prevalence of hospital vs. community acquired VTE over time. Methods. Using RAMQ medical service and prescription claims data, we identified a cohort of elderly persons with incident VTE between 1983 and 1994. All individuals 65 years of age and older between January 1, 1994 and December 31, 2004 with at least one medical service (i.e. physician) claim linked to a VTE ICD-9 code in conjunction with a prescription claim for an anticoagulant in the subsequent 60 days, and who had no prior VTE-coded claim between 1983 and 1994 were included in the cohort. The first (incident) VTE-coded claim (index claim) during the period between January 1, 1994 and December 31, 2004 defined entry into the cohort. Using Québec population census data, we determined annual VTE incidence estimates, adjusting for the population's age and sex distribution as per 1999 census data. An index VTE event that occurred during a hospitalization and up to 90 days following hospital discharge was defined as hospital-acquired VTE, otherwise it was considered to be community-acquired. Results. A total of 27 758 persons were included in our cohort. The age and sex adjusted annual VTE incidence among individuals 65 years of age and older was 2.1, 2.7, and 2.8 per 1000 population in 1994, 1999 and 2004, respectively (p trend 〈 0.001 (across years)). The incidence rates increased with age and were slightly higher in men than women. Overall, 35% of VTE events were hospital-acquired (n=9598) and 65% occurred in the community (n=18160). There was a significant trend over time of an increase in the proportion of VTE associated with hospitalization with 32% of VTE being hospital acquired in 1994 and 39% in 2004. Conclusion. In a general population of individuals 65 years of age and older, VTE is a common problem, and its overall annual incidence has increased over time. Our results suggest that hospital admission and recent hospitalization are increasingly important risk factors for VTE occurrence in this population, and that efforts are needed to improve and optimize VTE preventative strategies in the hospitalized elderly. Disclosures: No relevant conflicts of interest to declare.

Description: Although generalized T-cell activation is an important factor in chronic HIV disease pathogenesis, its role in primary infection remains poorly defined. To investigate the effect of immune activation on T-cell changes in subjects with early HIV infection, and to test the hypothesis that an immunologic activation “set point” is established early in the natural history of HIV disease, a prospective cohort of acutely infected adults was performed. The median density of CD38 molecules on CD4+ and CD8+ T cells was measured longitudinally in 68 antiretroviral-untreated individuals and 83 antiretroviral-treated individuals. At study entry, T-cell activation was positively associated with viremia, with CD8+ T-cell activation levels increasing exponentially at plasma HIV RNA levels more than 10 000 copies/mL. Among untreated patients, the level of CD8+ T-cell activation varied widely among individuals but often remained stable within a given individual. CD8+ T-cell activation and plasma HIV RNA levels over time were independently associated with the rate of CD4+ T-cell loss in untreated individuals. These data indicate that immunologic activation set point is established early in HIV infection, and that this set point determines the rate at which CD4+ T cells are lost over time.