ALBERT

Description: Cancer patients often have an activated clotting system and are at increased risk for venous thrombosis. In the present study, we analyzed tissue factor (TF) expression in 4 different human pancreatic tumor cell lines for the purpose of producing derivative tumors in vivo. We found that 2 of the lines expressed TF and released TF-positive microparticles (MPs) into the culture medium. The majority of TF protein in the culture medium was associated with MPs. Only TF-positive cell lines activated coagulation in nude mice, and this activation was abolished by an anti–human TF Ab. Of the 2 TF-positive lines, only one produced detectable levels of human MP TF activity in the plasma when grown orthotopically in nude mice. Surprisingly, 〈 5% of human TF protein in plasma from tumor-bearing mice was associated with MPs. Mice with TF-positive tumors and elevated levels of circulating TF-positive MPs had increased thrombosis in a saphenous vein model. In contrast, we observed no difference in thrombus weight between tumor-bearing and control mice in an inferior vena cava stenosis model. The results of the present study using a xenograft mouse model suggest that tumor TF activates coagulation, whereas TF on circulating MPs may trigger venous thrombosis.

Description: Introduction: The majority of published studies evaluating inhibitors have focused mainly on patients with severe hemophilia A. In non-severe hemophilia A (NSHA) patients, the development of inhibitors can have a profound clinical impact, with major bleeding complications similar to that of patients with severe or acquired hemophilia. Yet, epidemiological data on inhibitors in NSHA patients, specifically mortality, is scarce and currently limited to the European and Australian cohort [Eckhardt CL, et al. J Thromb Haemost. 2015 Jul;13(7):1217-251]. Objectives: To determine the all-cause and inhibitor-related mortality in NSHA patients in the United States using the ATHNdataset Methods: Subjects and study design The ATHNdataset is a 'limited dataset' as defined under the United States Health Insurance Portability and Accountability Act (HIPAA) to be free of protected health information, with data collection by more than 130 hemophilia treatment centers (HTC) across the United States. It includes patients with congenital bleeding disorders in the United States who have authorized the sharing of their demographic and clinical information for research. Data collection and definitions The ATHNdataset was queried on December 31, 2018 to extract the following information on NSHA patients: Patient demographics, inhibitor status, date of death, and primary cause of death. The presence of inhibitors was defined as: (i) ≥ 2 positive Bethesda inhibitor assay titers of ≥ 1.0 BU/mL; or (ii) a decrease in plasma FVIII coagulant activity (FVIII:C) to at least 50% of baseline activity and/or spontaneous bleeding symptoms in patients with inhibitor titers between 0.6 and 1.0 BU/mL. Patients who had a negative inhibitor history or have never been tested for FVIII inhibitors were classified as negative for inhibitors. Statistical analyses The person-year mortality rate was calculated as the ratio of the number of deaths to the number of person-years at risk, presented as rates per 1000 person-years. Person-years at risk was calculated for each patient as the time between the start of the observation period (January 1, 2010 or date of birth for patients who are born later) and the end of the observation period (date of death, loss-to follow-up or December 31, 2018). Patients who were deceased or lost to follow-up before January 1, 2010 were not included in the analysis. Inhibitor person-years at risk for inhibitor patients was calculated from January 1, 2010 if the first positive inhibitor test occurred prior to January 1, 2010 or from the date of the first positive inhibitor test that occurred during the observation period until the end of the observation period. Inhibitor-related death was attributed to all patients who had a positive inhibitor history. Mortality rates were compared between inhibitor and non-inhibitor patients using z- test. Results: Between 1/1/2010 and 12/31/2018, the ATHNdataset included 6,606 NSHA patients who were born between 1920 and 2018. Patients were observed for a total of 56,064 person-years. 85.57% (n = 5,653) of these patients were observed for the full nine years. The average follow-up time per patient was almost 8.5 years. Inhibitors developed in 171 (2.59%) NSHA patients. The median age for inhibitor development was 13 years (IQR, 6 - 37 years) and the mean age was 22 years. Demographics characteristics of the patients are listed in Table 1. All-cause mortality At the end of follow-up, there was a total of 136 deaths in the NSHA population, occurring at a median age of 63 years (IQR, 51 - 75 years). The overall all-cause mortality rate was 2.43 per 1,000 person-years (95% CI: 2.02 - 2.83). The most common primary cause of death was cancer (n=27, 19.9%) (Table 2). Inhibitor-related mortality Three deaths were associated with inhibitors. Inhibitor-related mortality rate was 2.40 per 1,000 person-years, whereas among the never inhibitor group, the mortality rate was 2.44 per 1,000 person-years (p = 0.790). Mortality risk ratio between inhibitor and never inhibitor was 0.98 (95% CI: 0.31 - 3.08). Conclusion: In NSHA patients, the development of inhibitors occurred at a relatively early age and was not associated with increased mortality. Disclosures Kempton: Novo Nordisk: Research Funding; Octapharma: Honoraria; Genentech: Honoraria; Spark Therapeutics: Honoraria. Key:Uniqure BV: Research Funding.

Description: Recent studies by our laboratory have demonstrated unequivocally that factor V is endocytosed by megakaryocytes from plasma to form the platelet-derived factor V pool. In the current study, we have determined the time-dependent acquisition of factor V by platelets in a 67 year old factor V-deficient patient, previously shown to be completely devoid of plasma- and platelet-derived factor V. The patient now receives weekly tranfusions of two units of FFP to prevent gastrointestinal bleeding. Plasma and platelet lysate samples were prepared from fresh, whole blood drawn prior to (0 hrs), and following (2, 6, 24, 96, 168 hrs) patient transfusion. A factor V radioimmunoassay (RIA), which can detect factor V concentrations as low as 0.075 μg/mL, a standard factor V clotting-based activity assay, and/or western blotting analyses, which utilize a mixture of anti-human factor V light chain and heavy chain mAbs, were used to evaluate the appearance, and subsequent disappearance, of factor V in these two blood compartments. Prior to transfusion (t = 0 hr), the patient’s plasma-derived factor V could not be detected by any of the three factor V assays. The patient’s plasma-derived factor V level peaked immediately following transfusion (t = 2 hr) reaching a concentration of 1.3 +/− 0.08 μg/mL as measured by RIA, and declined until it reached an undetectable level at 96 hrs post transfusion. These data were confirmed by the results of both the clotting-based assays and western blotting analyses. As the patient’s platelet-derived factor V levels were below the sensitivity of both the RIA and clotting assay, they were analyzed by western blotting. Such analyses confirmed that subsequent to its endocytosis, the patient’s platelet-derived factor V is stored in a partially protelytically activated form similar to that of a control individual. Due to the partially proteolytically activated state of the platelet-derived cofactor, platelet lysates treated with thrombin to activate the factor V to factor Va were used for quantative western blotting. Interestingly, and perhaps consistent with the patient’s receipt of weekly FFP transfusions, factor V/Va could still be detected in platelets immediately prior to transfusion. Subsequent to transfusion and in marked contrast to changes in the plasma-derived cofactor pool, a significant increase in the patient’s platelet-derived factor V/Va level was not observed until 24 hrs post transfusion. These data are consistent with previous studies demonstrating that platelets do not endocytose factor V. Although the concentration of the platelet-derived factor V/Va pool decreased over the subsequent 6 days, antigen remained detectable even though the plasma-derived pool had been depleted 3 days earlier. These combined observations indicate that, subsequent to FFP administration, the patient’s megakaryocytes acquire and proteolytically process plasma-derived factor V normally. Furthermore, the consistent presence of factor V/Va within the patient’s platelets is in all likelihood preventing gastrointestinal bleeding in this individual, which supports the concept that platelet-derived factor V represents the hemostatically relevant factor V pool.

Description: Blood microparticles (MPs) in sickle cell disease (SCD) are reportedly derived only from erythrocytes and platelets. Yet in SCD, endothelial cells and monocytes are activated and abnormally express tissue factor (TF). Thus, sickle blood might contain TF-positive MPs derived from these cells. With the use of flow cytometry to enumerate and characterize MPs, we found total MPs to be elevated in crisis (P = .0001) and steady state (P = .02) in subjects with sickle cell disease versus control subjects. These MPs were derived from erythrocytes, platelets, monocytes, and endothelial cells. Erythrocyte-derived MPs were elevated in sickle crisis (P = .0001) and steady state (P = .02) versus control subjects, as were monocyte-derived MPs (P = .0004 and P = .009, respectively). Endothelial and platelet-derived MPs were elevated in sickle crisis versus control subjects. Total TF-positive MPs were elevated in sickle crisis versus steady state (P = .004) and control subjects (P 〈 .0001) and were derived from both monocytes and endothelial cells. Sickle MPs shortened plasma-clotting time compared with control MPs, and a TF antibody partially inhibited this procoagulant activity. Markers of coagulation were elevated in patients with sickle cell disease versus control subjects and correlated with total MPs and TF-positive MPs (P 〈 .01 for both). These data support the concept that SCD is an inflammatory state with monocyte and endothelial activation and abnormal TF activity. (Blood. 2003;102:2678-2683)

Description: Abstract 1515 Poster Board I-538 BACKGROUND Acute chest syndrome (ACS) is a common cause of morbidity and mortality for individuals with sickle cell disease (SCD). The treatment of ACS is mainly supportive. Prior studies have shown that intravenous pulse-dose corticosteroids, such as dexamethasone, can decrease the duration and morbidity of ACS. However, such treatment may also precipitate ‘rebound‘ episodes of vaso-occlusive pain in some individuals that might negate the overall benefit of corticosteroids. Tapered corticosteroid therapy might decrease the rebound effect while maintaining therapeutic benefit. Therefore, we designed a prospective study to begin to test this hypothesis and to assess biomarkers that might clarify the therapeutic and toxic mechanisms of corticosteroids in SCD, predict outcome, and guide therapy. METHODS We conducted a multi-center, placebo-controlled pilot study to test the feasibility and safety of high-dose oral dexamethasone followed by a taper for ACS. Children and adults with SCD and ACS of any severity were randomized to dexamethasone (0.3 mg/kg q12h x 2, 0.3 mg/kg q24h x 2, 0.2 mg/kg q24h x 2, 0.1 mg/kg q24h x2, then stop) or placebo to start within 24 hours of diagnosis. All subjects received standard, protocol-directed supportive care for ACS. We defined the duration of ACS using a novel, objective tool that assessed rate and effort of breathing, oxygen saturation in room air, thoracic pain, and use of supplemental oxygen and ventilatory support. The primary outcomes were the duration of ACS and the duration of hospitalization for ACS. We also measured a panel of biomarkers before, during and after therapy. Inflammatory biomarkers included high sensitivity C-reactive protein and secretory phospholipase A2 (sPLA2). White cell and endothelial activation markers included sVCAM-1, sICAM-1, vWF:Ag and vWF:RCoF, sE-selectin, sP-selectin, sL-selectin, nitric oxide metabolites (NO), and whole blood tissue factor. We used generalized linear mixed models controlling for age to test for differences between treatment groups. RESULTS We enrolled 12 subjects (9 children, 3 adults; mean age 17.3 years, range 5 - 45) with homozygous sickle cell anemia at 4 centers and randomized 11 (1 drop-out) to either dexamethasone (N=5) or placebo (N=6). The objective ACS assessment tool was completed on all subjects without difficulty. In this pilot study, dexamethasone reduced the duration of hospitalization (41.5 vs 62.3 hrs; P=0.024), but not the duration of ACS (log of duration 2.4 vs 3.5 hrs; P=0.127), supplemental oxygen (17.5 vs 41.2 hrs; P=0.876), hypoxemia (13.8 vs 34.3 hrs; P=0.770), or total opioid usage in morphine equivalents (54.4 vs 68.8 mg; P=0.885). There were no statistically significant differences in adverse events between arms. However, 3 patients treated with dexamethasone had a painful event in the 2 weeks after hospital discharge (1 required re-hospitalization), compared to 1 patient in the placebo group (0 re-hospitalizations). No marked leukocytosis occurred in either treatment group, but the leukocyte count at 1-week follow-up had decreased less from baseline in the dexamethasone group compared to placebo (-17.7 vs -37.6%; P=0.028). The baseline sPLA2 concentration was 3 100 ng/mL in 4/5 and 4/6 patients in the dexamethasone and placebo arms. The white cell activation marker, sL-selectin was significantly decreased at 1-week follow-up in the dexamethasone group compared to placebo (573.8 ng/mL vs 742.8; change from baseline -121.2 vs 57.2; P

Description: Introduction Inhibitors are considered to be the most serious complication of clotting factor concentrate (CFC) therapy in hemophilia. The incidence of inhibitors in PTPs with severe hemophilia is very low (∼ 1 per 1,000 patient years). Four cases of PTP inhibitors that developed during hospital admissions were observed at the University of North Carolina over a 14-month period during 2011 – 2012. Due to concern about the possible role of the CFC used during hospitalization, further investigation by the CDC was requested. Methods A review of hemophilia-related hospital practices and procedures, along with a medical record review of admissions from January 2008 – November 2012 was conducted to: 1) compare inhibitor incidence trends in the time periods before and after January 2011; and 2) assess patient risk factors and evaluate hospital practices relative to inhibitor occurrence. A case was a person who developed an inhibitor during a hospital admission and non-cases were inpatients with no history of inhibitor development. The prevalence of inhibitor risk factors were compared between cases and non-cases and across time periods. Unadjusted odds ratios were used to compare the prevalence of exposures between cases and non-cases and comparisons of means of continuous risk factors used parametric and non-parametric tests as appropriate. Results A check of the FDA's Adverse Event Reporting System (FAERS) revealed no identified problems of the CFC in question. The medical record review revealed 134 admissions in 49 patients (59 in period 1 and 75 in period 2) during the 5-year period (see Figure). No other cases were found during the review; however, one of the index cases was found to have developed the inhibitor as an outpatient leaving 3 cases and 45 non-cases for study. The cases ranged in age from 23 – 69 years. One had severe and one had moderate hemophilia A, while the third case had combined factor V-VIII deficiency with FV and FVIII levels of 7% and 10%, respectively. Compared to non-cases, cases had elevated odds of: an infection (OR, 95% CL=4.4, 0.3–53), continuous factor infusion (CI) (4.2, 0.04–6.4), at least one non-surgical procedure (3.9, 0.1–80.5), and used the only hospital-available CFC (2.7, 0.1–73) during the admission and a product switch prior to admission (3.5, 0.1–105) and a family history of hemophilia (2.4, 0.1–51). In addition, cases had more hospital admissions (mean 6.6 vs. 2.4, p = 0.002) and more total hospital days (mean 39.6 days vs. 14.8 days. p = 0.05) than non-cases. Finally, cases received a greater total dose of CFC than non-cases (mean 101 IU/kg vs. 69 IU/kg, p = 0.003) during admission. Two hospital practices changed in period two: 1) the method of preparation and administration of CI involved less diluent and, therefore, a more concentrated infusion solution; and 2) the hospital pharmacy stocked only one brand of CFC in period 2 whereas several brands were available in period 1. The latter change increased the proportion of inpatients using a different product than that used as an outpatient (i.e., product switch) during period 2. Compared to inpatients admitted during period 1, those in period 2 were: more likely to receive the only available CFC and to have a product switch during the admission (p 〈 0.001). No difference between time periods was seen in the proportion of patients administered CI (58.8% vs. 56.9%) or who were tested for an inhibitor during admission (25% vs. 22%). Conclusions The investigation results support an increase in incidence of inhibitors among inpatients during period 2 compared to period 1, however, the number of cases was small. Nonetheless, because inhibitors are rare in PTPs, the occurrence of 3 cases in a 14-month period after a 3-year time span with no cases is highly suggestive of an actual increase in occurrence. The apparent increase was unlikely due to enhanced surveillance since rates of inhibitor testing during the two time periods were similar. While the odds ratios for some of the risk factors were elevated, the confidence limits were wide indicative of the lack of study power. Although there was no clear indication of a preventable inciting factor and more investigation is needed, the investigation revealed that this inhibitor cluster occurred in patients who had many complications that required lengthy hospitalizations and intense treatment with CFCs, contributing factors that should be considered in future management of hemophilia patients. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3326 Immune thrombocytopenia (ITP) is a relatively prevalent disease in dogs with significant morbidity and mortality. Canine ITP is clinically analogous to human ITP, with heterogeneity in bleeding manifestations in individuals with similar platelet counts. With a view to ultimately investigate this bleeding heterogeneity, we set out to develop a canine model of ITP. There are currently no existing large animal models of ITP. An induced canine ITP model would be representative of ITP without the confounding co-morbidities seen in clinical cases. Since spontaneous ITP occurs in both dogs and humans, the dog is an ideal translational model. We hypothesized that 2F9, a murine IgG2a monoclonal antibody to the canine platelet glycoprotein GPIIb (a common target of autoantibodies in ITP), would induce predictable dose-dependent thrombocytopenia (TCP) in healthy dogs. 2F9 had not been previously administered in vivo. We produced highly purified 2F9 and αYFA antibodies from the 2F9 hybridoma (gift of David Wilcox, Blood Research Institute, Wisconsin) and an isotype control murine anti-yellow fever antibody (αYFA) hybridoma. A dose titration (2 dogs) and a dose repeatability study (3 dogs) were performed in healthy adult research dogs by repeated intravenous infusion (≤ 6 doses) of 2F9 antibody until a target nadir of 5–30 × 103 platelets/μl was reached. Platelet counts were performed hourly until the platelet count reached the desired nadir range (t=0 hrs), after which complete blood counts were performed at 2, 4, 6, 8, 12, 24 hours, then q 24 hours for 10 days. The following were evaluated throughout the study: physical examination, buccal mucosal bleeding time (BMBT, baseline and t=0 only), serum cytokines and chemokines (INFγ, Interleukin (IL) 2, 6, 7, 8, 10, 15, 18, KC, IP-10, MCP-1, GM-CSF, TNFα; Milliplex CCYTOMAG-90K), fibrinogen, and D-dimers. Specificity of the 2F9 effect was confirmed by IV infusion of the isotype control (αYFA) to 3 dogs at the highest cumulative effective dose of 2F9 (167 μg/kg); all parameters were measured as above (t=0 hrs was one hour after αYFA dosing). Within 2 hours of a median cumulative 2F9 administration of 63 μg/kg (range 50.0–166.6 μg/kg), all dogs developed profound TCP (range 11–28 × 103/μl). Compared to the control group, platelet nadir was significantly lower (median (range): 6 (4–11) × 103/μl vs. 200 (179–209) × 103/μl; p= 0.036) and change in platelet count from baseline to nadir was significantly greater in the 2F9-treated group (median (range): 238 (179–325) × 103/μl vs. 4 (0–10) × 103/μl; p=0.036) (Fig 1); p-values were calculated using the exact Wilcoxon rank-sum test. Platelet nadir was in our target range and platelet count remained 〈 40 × 103/μl in all 2F9-treated dogs for 24 hours. Dosing was predictable: in each dog, after an initial dose of 50 μg/kg 2F9, the second dose needed to reach the target nadir could be accurately calculated from the initial platelet decrease. 2F9-treated dogs developed a range of clinical bleeding from none to petechiae, ecchymoses, melena, and hematuria. At t=0 hrs, BMBT increased 3–8 fold in treated dogs, compared to 〈 2 fold in control dogs. Dogs had no changes in vital signs or demeanor and did not require any transfusion support. The model does not appear pro-thrombotic as fibrinogen and D-dimers were similar over time in 2F9-treated vs. control dogs. 2F9 infusion also generated negligible systemic inflammation, as assessed by white blood cell count and serum cytokine measurement. Unexpectedly, however, serum IL8 tracked faithfully with platelet count, demonstrating that platelets are a major source of serum IL8 in dogs (Fig 2). Although α granules are known to contain IL8, platelets have not been previously described as a significant serum IL8 source. Since IL8 is an important neutrophil chemokine, our finding may illuminate a novel mechanism of platelet-neutrophil cross-talk. In summary, we have developed a novel large animal ITP model that is highly representative of the spontaneous disease. Like naturally-occurring ITP, dogs demonstrate bleeding heterogeneity despite similar platelet counts (data not shown). We expect our model to lead to further insights into bleeding mechanisms in ITP. Ultimately, understanding what factors predispose certain patients to bleed will allow us to exploit these factors therapeutically as novel ITP treatments. Disclosures: No relevant conflicts of interest to declare.

Description: Background: Compared to Whites, Black-Americans may have a 40% higher incidence of idiopathic VTE. However, whether other VTE characteristics vary by race is uncertain. Objective: To compare demographic and baseline characteristics among White- and Black-Americans with VTE. Methods: Using a standardized data-collection form, demographic and baseline characteristics were prospectively collected from consecutive consenting patients enrolled in seven Thrombosis and Hemostasis Centers from August 2003 to March 2008. For patients with objectively diagnosed VTE, demographic and baseline characteristics were compared among White- and Black-American VTE patients, both overall, and by age and gender. Results: Among 1960 White- and 368 Black-Americans with VTE, compared to Whites, Blacks had significantly less isolated DVT (73.9% vs. 86.5%, p