ALBERT

Description: Abstract 701 Diadenosine triphosphate (Ap3A) is a component of platelet dense granules, and is released into the extracellular space during the second wave of platelet aggregation following platelet activation by ADP and other agonists. Ap3A has long been thought to stimulate platelet aggregation after release into the extracellular space by providing a prolonged source of ADP via hydrolysis by a slow extracellular enzyme present in human plasma. Here, we identify NPP-4, a member of the nucleotide pyrophosphatase/phosphodiesterase enzyme family present on endothelial cell surfaces, as a potent hydrolase of Ap3A capable of stimulating platelet aggregation and secretion at nanomolar concentrations. We performed lumiaggregometry with citrated platelet-rich plasma in the presence of Ap3A with or without increasing nanomolar concentrations of NPP-4. In these experiments we determined that Ap3A alone in concentrations up to 80 uM initiated a primary wave of platelet aggregation that was followed by rapid disaggregation. In the presence of nanomolar concentrations of NPP-4, however, the primary and secondary waves of platelet aggregation and dense granule release are strongly induced in an enzyme concentration-dependent fashion. In contrast, mutant NPP-4, catalytically inactivated by an active site threonine to alanine mutation, had no effect on platelet aggregation and dense granule release under identical conditions. In order to clarify the structural basis of the enzymatic mechanism, we determined the high-resolution structure of NPP4 in the presence and absence of the enzymatic product, AMP. In aggregate, our studies define the biological, enzymatic, and molecular basis for NPP-4 and Ap3A activity in platelet aggregation and secretion in vitro and suggest that NPP-4 may play a role in hemostasis in vivo by augmenting platelet aggregation and release of granule contents at the site of vascular injury. With these studies we have thus established the molecular foundation for the rational development of a new class of therapeutics capable of modulating vascular thrombosis. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 5100 Two male first cousins with mild hemophilia A had symptomatic electrocardiographically documented paroxysmal atrial fibrillation for several years that had become resistant to pharmacologic suppression. Both had baseline factor VIII levels of 12 to 15% and experienced bleeding requiring coagulation factor infusion therapy in response to trauma and surgical procedures. Radiofrequency ablation of ectopic electrical foci was considered in both cases but deferred because anticoagulation required to prevent thromboembolic complications of radiofrequency ablation carried an unacceptably high risk of bleeding. Iron accumulation with aging has been implicated in the pathogenesis of cardiovascular disease in general and arrhythmias in particular. Uncompensated iron-catalyzed oxygen free radical formation accounts for such toxicity. Remission of arrhythmias has been reported with iron reduction therapy in patients with iron overload disorders. Effects of iron reduction on arrhythmias in settings other than iron overload syndromes are unknown. Body iron stores as assessed by serum ferritin levels were elevated in both men (389 and 305 ng/ml respectively) but neither had the C282Y or H63D genes for hemochromatosis. Calibrated reduction of iron stores by serial phlebotomy, avoiding iron deficiency, was performed and was followed by sustained remission of PAF in both cases. These findings suggest that iron reduction with maintenance of ferritin levels below about 100 ng/ml may be effective treatment for arrhythmias apart from the classic iron overload syndromes. Effects of primary prevention of iron accumulation over time and reduction of existing elevated iron stores deserve further study in patients with bleeding disorders in whom standard treatment of diseases of aging may be costly and carry high risk. The efficacy, safety, simplicity and cost effectiveness of iron reduction therapy commends application of this investigational approach to disease prevention and treatment in the general population. Disclosures No relevant conflicts of interest to declare.

Description: Introduction Myelodysplastic syndromes (MDS) comprise a heterogeneous group of hematopoietic stem cell disorders characterized by bone marrow failure and dysplasia of one or more blood cell lineages resulting in peripheral blood cytopenias with frequent progression to AML. Treatment with azanucleosides improves survival and quality of life without the toxicities associated with intensive chemotherapy programs. The azanucleosides are DNA demethylating agents that may improve hematopoiesis in MDS by releasing tumor suppressor genes from silencing due to promoter hypermethylation. MicroRNAs (miRNAs) play key roles in cell growth and differentiation and oncogenic transformation. Dysregulation of miRNA expression may contribute to the development of MDS, as expression of several miRNAs has been reported to be downregulated in bone marrow cells from MDS patients. We performed the current study to test the hypothesis that treatment with demethylating agents modulates miRNA expression in hematopoietic tissue from MDS patients and to characterize the specific changes in miRNA expression. Methods This was a two-center, prospective cohort study to evaluate miRNA expression signatures in hematopoietic progenitor cells isolated from the bone marrow of subjects undergoing evaluation for peripheral blood cytopenias. Subjects were included for analysis if RNA of sufficient quality was available and the final pathologic diagnosis was one of the following: 1) normal (served as controls), 2) dyspoiesis without meeting criteria for MDS and without cytogenetic abnormalities, 3) MDS and 4) acute myeloid leukemia (AML) with myelodysplasia-related changes. Subjects who underwent treatment with a demethylating agent had bone marrow collected after treatment for before and after comparisons of miRNA signatures. Mononuclear cells were isolated from bone marrow aspirate specimens by density gradient centrifugation using a Ficoll-Paque technique. MicroRNA was isolated using the miRNeasy kit (Qiagen) and was analyzed using miScript miRNA PCR Array Human Cell Development & Differentiation (MIHS-103Z, Qiagen). Isolated microRNA was checked for quality control using miScript miRNA QC PCR array. Data were analyzed using Qiagen analysis software for microRNA, and miRNA levels are expressed as 2-ΔΔCT. P-values 2 fold decrease in 22/84 miRNAs), most notably miR-126-3p (p = 0.02) and miR-20a-5p (p = 0.04). In contrast, high-risk MDS patients demonstrated a significant overall increase in microRNA expression (〉2-fold increase in 33/84 miRNAs), especially miR-20b-5p (p = 0.04) and miR-125b-5p (p = 0.01). Treatment with demethylating agents led to decreased expression of most miRNAs (〉2-fold decrease in 14/84 miRNAs), including miR-181a and let-7a, but an increase in others, such as miR-22 (2.3-fold increase). We obtained similar results in experiments conducted with primary bone marrow samples that were treated with demethylating agents ex vivo. Summary Our study is the first to characterize the changes in miRNA expression patterns in patients with MDS treated with demethylating agents. We also identify novel miRNA candidates to be further evaluated as biomarkers for assessing response to treatment with demethylating agents (e.g., miR-22, which has been described as possibly having a role in regulating TET2). We also demonstrate the striking difference in the pattern of miRNA expression between low-risk and high-risk MDS patients, a feature that may one day be exploited to assist with making treatment decisions. Disclosures: No relevant conflicts of interest to declare.

Description: Background: Patients undergoing bariatric surgery are at increased risk for symptomatic venous thromboembolism (VTE). Although the incidence of clinical VTE is lower than in other general and orthopedic surgical procedures, pulmonary embolism is an independent predictor of death after gastric bypass surgery. The optimal strategy for postoperative thrombophylaxis in bariatric surgery has yet to be elucidated. In 2010 our institution proposed a guideline for postoperative thrombophylaxis for bariatric surgery based on risk stratification. Patients were considered high risk if they had history of VTE or a BMI 〉/= 60 kg/m2 or if they had two or more of the following: age 〉 50, BMI 〉/= 50 kg/m2, male sex, recent history of smoking, obstructive sleep apnea, varicose veins, or hormone therapy within 30 days. All patients undergoing bariatric surgery received enoxaparin 40 mg SC twice daily in addition to mechanical thrombophylaxis including venodynes and compression stockings during the hospital stay. Prophylactic IVC filter insertion was not recommended. High-risk patients received an extended course of anticoagulation for 10 days after discharge. Methods: We retrospectively reviewed the medical charts of 692 patients who underwent bariatric surgery at Dartmouth-Hitchcock Medical Center from 2009 to 2014. We analyzed the incidence of VTE and bleeding complications under two different institutional thrombophylaxis protocols: an earlier protocol of VTE prophylaxis based on surgeon's preference (n = 62) and our subsequent protocol based on risk stratification (n = 630). Results: Prior to implementing our protocol the incidence of VTE and bleeding complications in patients who underwent bariatric surgery was 1.6% (1 in 62) for each. After 2010, with the implementation of extended VTE prophylaxis for high-risk patients, the incidence of postoperative VTE was 0% (0 in 630) and the incidence of postoperative bleeding was 2.7% (17 in 630). Of 17 patients who developed postoperative bleeding, 14 (82%) developed bleeding before postoperative day 3 during the hospitalization and 3 (18%) experienced delayed bleeding after hospital discharge. Of 3 patients with delayed bleeding, only 1 was on the extended thrombophylaxis protocol. Severe bleeding, defined as the need for packed red blood cell transfusions or re-operation, occurred in 1.6% (10 in 630). Conclusion: Our study demonstrates that a protocol based on risk stratification is effective at reducing the risk of symptomatic VTE in patients undergoing bariatric surgery. Although the incidence of all postoperative bleeding appears to have increased slightly after implementation of the protocol, the incidence of severe bleeding appears unchanged. As most of the post-operative bleeding events occurred during the hospitalization period, extending the length of pharmacologic thromboprophylaxis was not associated with an increase in bleeding. This study is limited by its retrospective design and the small number of patients studied prior to implementing the extended VTE prophylaxis protocol. Nevertheless, the findings are promising with respect to VTE risk reduction after bariatric surgery and suggest that prospective evaluation of the thromboprophylaxis protocol is in order. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 3143 Background: AHA is a rare bleeding disorder caused by autoantibodies (inhibitors) to coagulation factor VIII (FVIII). Acquired FVIII inhibitors are associated with a high rate of complications, with major bleeding reported in up to 90% of affected individuals and mortality rates as high as 22%. There are currently no formal guidelines for the treatment of AHA, due to the paucity of data from randomized clinical trials and limited number of cases. However, there is general agreement that treatment should consist of a two-pronged approach that includes 1) achieving hemostasis in bleeding patients by administering FVIII bypassing agents, and 2) reducing or eradicating the inhibitor with immunosuppressive treatments such as rituximab (R), cyclophosphamide (C), prednisone (P) or a combination. C and P have been used as first-line treatment for AHA until the late 1990s, when R began to be used off-label for inhibitor eradication. Data from case reports and series suggest that R is effective as both first and second-line therapy for AHA. We hypothesized that the use of R rather than C and P may be cost-effective by reducing the overall use of expensive hemostatic agents. The purpose of this study was to compare clinical course, use of hemostatic agents and costs in patients who received R to historical patients who did not receive R for treatment of AHA. Methods: We performed a retrospective study of patients with AHA, defined by prolonged aPTT (〉37 sec), FVIII level 0.40 Bethesda Unit (BU)) at Dartmouth-Hitchcock Medical Center (DHMC) using the hospital's electronic medical record system, CIS, to collect pertinent demographic and clinical information. The data collection variables included: age, sex, co-morbidities, FVIII levels, FVIII antibody titers, type of bleeding, amount and total acquisition cost of hemostatic products administered, immunosuppressive regimens administered, time to complete remission (CR) (defined as FVIII level 〉 30%), and relapses. Results: We identified 16 patients with AHA treated at DHMC from 4/1997 to 3/2010. The median age at diagnosis was 78 (range 66 to 93), and 56% were women. Co-morbidities included current or prior malignancy (62%) and autoimmune disease (12%). Idiopathic cases accounted for 32%. One patient had both a malignancy and autoimmune disease. The median aPTT at diagnosis was 80 sec (range 53 to 〉160 sec); FVIII level, 3% (range

Description: Introduction: Management of VTE in patients with hematological malignancies is challenging as these patients often have thrombocytopenia and are at increased risk for bleeding. The International Society of Thrombosis and Hemostasis (ISTH) guidance statement recommends adjusting anticoagulation based on the severity and acuity of VTE and considering platelet transfusion support to maintain platelet counts above 〉50K/µL to facilitate full-dose anticoagulation (AC) in patients with acute VTE with high-risk features. For acute VTE with low-risk features, a dose-modified strategy such as half- or prophylactic-dose AC, depending on platelet count, is recommended. While the risk of AC with supportive platelet transfusions may be justified in patients with severe VTE, lower-dose AC without platelet transfusion support may be adequate for patients with non-severe VTE and may reduce the risk for development of platelet alloantibodies. To better understand practice patterns at our institution, we reviewed the management of VTE in patients with acute leukemia over an 8-year period. Methods: We retrospectively reviewed all patients with acute leukemia who developed acute VTE during induction chemotherapy at Dartmouth-Hitchcock Medical Center for the 8-year period (2006-2013). Primary outcomes were 1) objectively confirmed, recurrent symptomatic VTE, and 2) clinically significant bleeding within 1 month after diagnosis of the index VTE. Secondary outcomes included IVC filter use and platelet alloantibody development within 3 months after diagnosis of VTE. We classified VTE events as acute or subacute/chronic and severe or non-severe. An acute event was defined as a new VTE diagnosed during the index hospitalization, while a subacute/chronic event was defined as a VTE that occurred prior to the index hospitalization for which the patient was being treated with AC during the hospitalization. A severe VTE was defined as a DVT involving femoral or more proximal vein segments, the SVC, cerebral veins or any PE. Non-severe events included UE DVT and distal DVT. We classified treatment at the time of diagnosis of VTE into one of the following categories: 1) full-dose AC with platelet transfusion to maintain platelet count 〉25-50K/µL, 2) half-dose AC with platelet transfusion to maintain platelet count 〉25-50K/µL, 3) prophylactic-dose AC without additional platelet transfusion beyond the standard of care, 4) no anticoagulation. We recorded the number of IVC filters inserted in each category. Descriptive statistics, including mean and standard deviation for continuous variables, and percentage and frequency for categorical variables, were calculated. Results: 40 subjects met entry criteria (55% male) with median age of 57.5 years. The majority (88%) had acute myeloid leukemia. Acute VTE was identified in 80% and VTE was severe in 55%. The degree of thrombocytopenia at the time of VTE diagnosis was 50K/µL (32.5%). Subjects were initially treated with full-dose AC (60%), half-dose AC (5%), prophylactic-dose AC (12%) or no AC (22.5%). An IVC filter was inserted in 10 subjects (25%), and five subjects (12%) developed platelet alloantibodies. Seventeen subjects (42%) experienced bleeding complications within the first month of VTE diagnosis, six of which (35%) were clinically significant. Half of these were on full-dose AC, and half underwent insertion of an IVC filter. Two subjects (5%) had VTE recurrence within 1 month, and in both cases, AC had been discontinued prior to the recurrence due to bleeding complications. Conclusion: We found that management of VTE in patients with acute leukemia and thrombocytopenia varies and that the risk for bleeding complications was high irrespective of AC dose. We also uncovered the problem of VTE recurrence due to premature discontinuation of AC as a result of bleeding and observed a surprisingly high incidence of platelet alloimmunization. Dose-modified AC without platelet transfusion support may be an attractive alternative for treatment of VTE in this population, but further study is required to balance bleeding and thrombosis risk. Disclosures No relevant conflicts of interest to declare.

Description: Background: The incidence of upper extremity deep vein thrombosis (UEDVT) is increasing due to the increased use of central venous catheters. In contrast to lower extremity DVT (LEDVT), there is limited data to guide management, with many current management recommendations based on extrapolation from LEDVT studies. The aim of this study is to evaluate the characteristics and mortality associated with hospitalized patients with UEDVT from a large national database. Methods: Using the 2012 National Inpatient Sample (NIS), admissions with the International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9-CM) code for UEDVT (453.82) were extracted, and data collected on age, sex, length of stay, mortality, and associated diagnoses and procedures. LEDVT (ICD-9-CM 453.40) data were extracted for comparison. Collected data were used to calculate odds ratios with corresponding 95% confidence intervals and p-values to show associations where applicable. Results: Of the 7,296,968 unweighted admissions in the 2012 NIS, 8,350 were associated with UEDVT, and 18,194 were associated with LEDVT (prevalence of 0.11% vs 0.25%, respectively). UEDVT and LEDVT rates were similar in men and women. However, compared to LEDVT, patients with UEDVT were younger (61yr vs. 67 yr; p

Description: Introduction AL amyloidosis, precipitated by the deposition of immunoglobulin light chains in various organs, is generally secondary to the benign or malignant proliferation of clonal plasma cells. Reviews from large amyloid centers show that approximately 2-3% of AL amyloidosis is associated with an underlying non-Hodgkin lymphoma (NHL). Of the non-Hodgkin lymphomas, lymphoplasmacytic lymphoma (LPL) is most commonly associated with systemic AL amyloidosis. Systemic NHL-associated AL amyloidosis tends to feature a high level of IgM paraproteinemia and a predilection for several different organ tropisms. Here we report the unique case of a 71-year-old male with an insidious course of hypotension and worsening renal failure of unknown etiology, who was ultimately diagnosed post-mortem with AL amyloidosis associated with an intravascular large B-cell lymphoma (IVLBCL). Case Description A 71-year-old white male presented to the hospital after a fall. His medical history included type II diabetes, spinal stenosis, and a 30-pack-year smoking history. Over the past few months, he noticed a steady decline in functional status. On admission, he was hypotensive with bilateral 2+ pitting lower extremity edema. Laboratory evaluation revealed leukocytosis, anemia, and acute kidney injury with elevated creatinine and blood urea nitrogen. Urinalysis showed 30 mg/dL of protein, along with the presence of white and red blood cells and moderate bacteria. Enterococcus was subsequently cultured from the urine. A chest X-ray revealed small bilateral effusions. The patient was treated for urosepsis with antibiotics and vasopressor support in the intensive care unit. His hypotension improved initially, but over the next 3 weeks his course was complicated by recurrent episodes of hypotension, altered mental status, and worsening renal function. An exhaustive laboratory workup was negative for infection, and a transthoracic echocardiogram showed no significant findings. Further evaluation included a normal cortisol stimulation test and TSH level, but a serum protein electrophoresis showed 0.13 g/dL of monoclonal IgM lambda immunoglobulin (Figure A) with a urine protein electrophoresis showing a low level of lambda free light chains. Abdominal fat pad biopsy was negative for amyloid. The patient's condition continued to deteriorate, and he died on hospital day 29 with no clear diagnosis. Post-mortem examination revealed a CD20+, lambda restricted intravascular diffuse large B-cell lymphoma involving the kidneys, prostate, a cecal polyp, and lungs (Figure B). Sections of lung tissue revealed scattered areas of extracellular eosinophilic material that stained brick red with Congo red staining (Figure C) and displayed apple green birefringence under polarized light (Figure D). Similar findings in the heart and kidneys were consistent with systemic amyloidosis. Bone marrow analysis showed normal trilineage hematopoiesis with no evidence for involvement by lymphoma or plasma cell neoplasm. Discussion This case exhibits the first reported association of two rare and diagnostically challenging disorders: intravascular large B-cell lymphoma (IVLBCL) and systemic AL amyloidosis. Patients with IVLBCL often present with nonspecific symptoms secondary to small vessel occlusion and subacute deterioration in performance status, as was the case with our patient. Our patient's underlying intravascular NHL with systemic AL amyloidosis proved difficult to diagnose with his low-level IgM paraproteinemia serving as the only clue to the cause of his illness. The negative fat pad biopsy added to the diagnostic difficulty, but was not surprising due to the low sensitivity of the test in detecting systemic amyloidosis. In addition, diagnosing intravascular lymphoma is difficult as patients present with nonspecific findings and often without lymphadenopathy, making post-mortem diagnosis common. Thus, this case sets a precedent for intravascular diffuse large B-cell lymphoma provoking the onset of systemic AL amyloidosis – a covert, morbid, and diagnostically challenging combination. Disclosures: No relevant conflicts of interest to declare.

Description: Background: Perioperative warfarin management is a clinical challenge as it requires careful assessment of both thrombosis and bleeding risk associated with the planned procedure. In our experience, many clinicians favor bridging over not bridging and often use full dose unfractionated or low molecular weight heparin even when a prophylactic dose would be a reasonable alternative. To improve and standardize the complex decision-making process for anticoagulation management for patients undergoing elective surgery we introduced an evidenced-based perioperative bridging anticoagulation protocol in 2017 based on the 9th edition of American College of Chest Physicians guidelines. The aim of the study was to investigate the impact of the perioperative bridging anticoagulation protocol on postoperative bleeding and thrombotic events in patients undergoing elective surgery at our institution. We hypothesized that adherence to the proposed protocol reduces the incidence of postoperative bleeding complications without an increase in thrombotic risk. Methods: We performed a retrospective chart review for all surgical inpatients who were on long-term anticoagulation with warfarin and who required interruption of warfarin around surgery at Dartmouth-Hitchcock Medical Center from June 2016, when the protocol was introduced, to June 2019. Data were extracted from the electronic medical record for each study subject and included demographics, clinical and radiologic data, indication for long-term anticoagulation (atrial fibrillation, venous thromboembolism, or mechanical heart valve), risk categories for thrombotic events, type of surgery, anticoagulation management before and after surgery including type and dose of short-acting anticoagulation used for bridging, and bleeding and thrombotic complications within 30 days of surgery. We evaluated adherence to the bridging protocol by individual providers and divided subjects into two groups for comparison: 1) subjects who received protocol-directed care and 2) subjects who received non-protocol care. We compared the risk of postoperative bleeding and thrombotic events in the two groups using the R3.5.1. Concordance of bridge protocol and crude associations between discrete variables were assessed by McNemar's and two-tailed Fisher's exact test, respectively. Multivariate logistic regression was used to estimate covariate-adjusted association groups and the outcomes of interest. P 〈 0.05 was used as the criterion for statistical significance. Results: 194 subjects met entry criteria and were included in the study; 114 subjects were managed according to the protocol (58.8%; 95% CI = 51.5%-65.8%, McNemar's P = 0.034). Clinical characteristics were similar in the protocol-adherent and nonadherent groups. Most patients (50%) were on long-term warfarin for stroke prevention with atrial fibrillation. The incidence of bleeding and thrombotic complications for the entire population was 7.8% and 4.7 %, respectively. Two-thirds of subjects who experienced either bleeding (66.7% vs 33.3%, 95% CI= 0.91-11.93, P = 0.055, two tailed Fischer's exact test) or thrombotic (66.7% vs 33.3%, 95% CI= 0.62-19.19, P = 0.16, two tailed Fischer's exact test) complications were in the protocol-nonadherent group. Controlling for age and sex using multivariate logistic regression, subjects whose anticoagulation was not managed postoperatively in adherence to the protocol were over three times as likely to experience a bleeding complication (adjusted odds ratio=3.36, 95% CI= 1.13-11.40, P = 0.036) and a thrombotic complication (adjusted odds ratio=3.54, 95% CI=0.88-17.77, P=0.088) when compared to patients whose postoperative anticoagulation management was protocol-driven. Discussion: Overall adherence to an evidence-based anticoagulation bridging protocol by providers was only 59%. When adhered to, protocol-based practice helped to reduce bleeding complications by 60% and was associated with a trend toward a lower risk for thrombosis. Our study results emphasize the value of an evidence-based perioperative anticoagulation bridging protocol and the importance of protocol adherence. To improve adherence, we have now embedded the protocol as an order-set in our electronic medical record system and have provided additional education to providers within our institution with a plan to assess improvement in adherence subsequently. Disclosures No relevant conflicts of interest to declare.

Description: Ap3A is a platelet-dense granule component released into the extracellular space during the second wave of platelet aggregation on activation. Here, we identify an uncharacterized enzyme, nucleotide pyrophosphatase/phosphodiesterase-4 (NPP4), as a potent hydrolase of Ap3A capable of stimulating platelet aggregation and secretion. We demonstrate that NPP4 is present on the surface of vascular endothelium, where it hydrolyzes Ap3A into AMP and ADP, and Ap4A into AMP and ATP. Platelet aggregation assays with citrated platelet-rich plasma reveal that the primary and secondary waves of aggregation and dense granule release are strongly induced by nanomolar NPP4 in a concentration-dependent manner in the presence of Ap3A, while Ap3A alone initiates a primary wave of aggregation followed by rapid disaggregation. NPP2 and an active site NPP4 mutant, neither of which appreciably hydrolyzes Ap3A, have no effect on platelet aggregation and secretion. Finally, by using ADP receptor blockade we confirm that NPP4 mediates platelet aggregation via release of ADP from Ap3A and activation of ADP receptors. Collectively, these studies define the biologic and enzymatic basis for NPP4 and Ap3A activity in platelet aggregation in vitro and suggest that NPP4 promotes hemostasis in vivo by augmenting ADP-mediated platelet aggregation at the site of vascular injury.