ALBERT

Description: Sudden infant death syndrome (SIDS), the leading cause of postneonatal infant mortality, likely comprises heterogeneous disorders with the common phenotype of sudden death without explanation upon postmortem investigation. Previously, we reported that ∼40% of SIDS deaths are associated with abnormalities in serotonin (5-hydroxytryptamine, 5-HT) in regions of the brainstem critical in homeostatic regulation. Here we tested the hypothesis that SIDS is associated with an alteration in serum 5-HT levels. Serum 5-HT, adjusted for postconceptional age, was significantly elevated (95%) in SIDS infants (n = 61) compared with autopsied controls (n = 15) [SIDS, 177.2 ± 15.1 (mean ± SE) ng/mL versus controls, 91.1 ± 30.6 ng/mL] (P = 0.014), as determined by ELISA. This increase was validated using high-performance liquid chromatography. Thirty-one percent (19/61) of SIDS cases had 5-HT levels greater than 2 SDs above the mean of the controls, thus defining a subset of SIDS cases with elevated 5-HT. There was no association between genotypes of the serotonin transporter promoter region polymorphism and serum 5-HT level. This study demonstrates that SIDS is associated with peripheral abnormalities in the 5-HT pathway. High serum 5-HT may serve as a potential forensic biomarker in autopsied infants with SIDS with serotonergic defects.

Description: Introduction Patients with Wiskott-Aldrich syndrome (WAS) including X-linked thrombocytopenia (XLT) have microthrombocytopenia, and hemorrhage is a major problem. Current management options in WAS/XLT patients include splenectomy, human stem cell transplant (HSCT) and gene therapy. In this study, we asked whether eltrombopag, a thrombopoietin mimetic, would increase platelet counts, improve platelet function, and/or reduce bleeding in WAS/XLT patients. Methods In 9 WAS/XLT patients and 8 age-matched healthy control subjects, flow cytometry was used to assess platelet function by surface expression of activated GPIIb-IIIa (reported by PAC1) and P-selectin in whole blood after stimulation with low and high concentrations of ADP or thrombin receptor activating peptide (TRAP), and by annexin V binding (a measure of surface phosphatidylserine) in platelet-rich plasma after stimulation with convulxin. Eltrombopag was administered to 5 WAS and 3 XLT patients (50 mg in 2 adults, and 1 mg/kg in 6 children up to 75 mg/day) with a goal platelet count ≥50k. Results High concentration ADP- or TRAP-induced PAC1 mean fluorescence intensity (MFI) was significantly reduced in WAS/XLT patients compared to healthy controls (Figure). Platelet surface P-selectin MFI in response to TRAP was also significantly reduced. In contrast, annexin V binding to platelets was not different between WAS/XLT and controls. As expected, platelet size of WAS/XLT patients was smaller than controls. WAS protein (which is deficient in WAS/XLT), is important for cytoskeletal movement and could therefore be involved in trafficking of surface proteins. However, surface expression of activated GPIIb-IIIa and P-selectin were no longer different in WAS/XLT patients vs. controls when corrected for size by platelet surface CD41 MFI. In 3 WAS/XLT patients whose platelet count improved on eltrombopag, platelet function did not improve. The table summarizes the results of eltrombopag treatment in 5 responders (2 WAS, 3 XLT patients) and 3 non-responders (3 WAS patients). Comparison of baseline, peak and change in immature platelet fraction in 5 WAS/XLT responders to eltrombopag vs. 7 pediatric chronic immune thrombocytopenia (ITP) patients responding to eltrombopag showed a significant decrease in all three measures, suggesting that platelet production in WAS/XLT patients is more difficult to increase than in ITP patients. Long term eltrombopag use in WAS/XLT patients showed no tachyphylaxis, transaminitis or induction of malignancy. Conclusions 1) Baseline platelet function in WAS/XLT is reduced compared to healthy age-matched controls, as measured by agonist-induced platelet surface activated GPIIb-IIIa and P-selectin. 2) This reduction is proportional to the reduced platelet size in WAS/XLT compared to controls. 3) In contrast, annexin V binding (a measure of platelet procoagulant activity) showed no differences between WAS/XLT and controls. 4) Eltrombopag has beneficial effects on the thrombocytopenia and bleeding, but not platelet function, in the majority of WAS/XLT patients. 5) This eltrombopag-induced reduction in bleeding is presumably primarily the result of the increased platelet count, but it was also observed in 2 eltrombopag “non-responders” (i.e. patients whose platelet counts did not increase after eltrombopag). 6) The production of new platelets with eltrombopag is less in WAS/XLT than in ITP. Disclosures: Off Label Use: Eltrombopag was given to WAS/XLT patients for treatment of thrombocytopenia. Michelson:Sysmex: Honoraria. Bussel:GlaxoSmithKline: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Amgen: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Cangene: Research Funding; Genzyme: Research Funding; IgG of America: Research Funding; Immunomedics: Research Funding; Ligand: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Eisai: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Shionogi: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Sysmex: Research Funding; Symphogen: Membership on an entity’s Board of Directors or advisory committees.

Description: The effects of eltrombopag, a thrombopoietin-receptor agonist, on platelet function in immune thrombocytopenia (ITP) are not fully characterized. This study used whole blood flow cytometry to examine platelet function in 20 patients receiving eltrombopag treatment at days 0, 7, and 28. Platelet surface expression of activated GPIIb/IIIa, P-selectin, and GPIb was measured with and without low and high adenosine diphosphate (ADP) and thrombin receptor activating peptide (TRAP) concentrations. Before eltrombopag treatment with no ex vivo agonist, platelet activation was higher in ITP patients than controls. Platelet GPIb and activated GPIIb/IIIa expression without added agonist was unchanged following eltrombopag treatment, whereas a slight increase in P-selectin was observed. Expression of P-selectin and activated GPIIb/IIIa in response to high-dose ADP was lower during eltrombopag treatment than at baseline. Eltrombopag led to a slight increase in platelet reactivity to TRAP only in responders to eltrombopag but not to levels above those in controls; whole blood experiments demonstrated that this increase was probably because of higher platelet counts rather than higher platelet reactivity. In conclusion, although thrombocytopenic ITP patients have higher baseline platelet activation than controls, eltrombopag did not cause platelet activation or hyper-reactivity, irrespective of whether the platelet count increased.

Description: Abstract 2141 Introduction: In patients with sickle cell disease (SCD), erythrocytes contribute to microvessel occlusion resulting in tissue damage and platelet activation. Platelet activation, aggregation, local thrombus formation and platelet activation-dependent leukocyte recruitment potentially amplify tissue ischemia. Antiplatelet therapy may therefore be useful in SCD. Here we evaluate levels of platelet activation markers in adolescents with SCD vs. normal controls and the effect of in vitro blockade of the platelet ADP receptor P2Y12 by prasugrel's active metabolite, R-138727. Methods: Blood was obtained from adolescents (10 – 18 yr) with SCD and healthy adult subjects. Platelet function was evaluated by: light transmission aggregation (LTA) in platelet-rich plasma with 20 μM ADP and in whole blood by VerifyNow P2Y12; Multiple Electrode Aggregometry (MEA) with 6.5 μM ADP; vasodilator stimulated phosphoprotein (VASP) P2Y12 assay; and whole blood flow cytometric analysis of basal and in vitro ADP-stimulated levels of platelet surface activated GPIIb-IIIa (reported by monoclonal antibody PAC1) and P-selectin, platelet-monocyte aggregates (PMA) and platelet-neutrophil aggregates (PNA). These endpoints were also evaluated after in vitro incubation of whole blood with R-138727 (0.1 – 10 μM). Results: In SCD patients compared with normal subjects, circulating PMA and PNA levels were significantly higher (76.5 ± 20.3% and 55.1 ± 21.8% vs. 20.1 ± 7% and 13.9 ± 4.2% [mean ± SD], respectively, p

Description: Background: The occurrence of thrombotic events despite aspirin (ASA) therapy in patients with vascular disease has been termed ASA resistance. However, limited data are available correlating laboratory evidence of platelet function with clinical ASA resistance. The PFA-100 collagen/epinephrine (COL/EPI) cartridge can detect the ASA-induced inhibition of platelet cyclooxygenase 1 in a high shear flow environment that mimics in vivo arterial conditions. Aim: To investigate whether there is an association of PFA-100 test results with clinical ASA resistance. Methods: Peripheral blood was collected prior to angiography from 560 sequential ASA-treated patients (81 or 325 mg 〉 3 d). Patients receiving thienopyridines were not excluded but those receiving GPIIb-IIIa antagonists were. Patients with normal angiographic arteries ("No CAD", low probability of ASA resistance) were compared to those with angiographic CAD or a single prior CAD event and to those with multiple CAD events ("CAD" and "Repeat CAD" respectively, higher probability of ASA resistance). Citrate 3.8% anticoagulated blood was tested in the PFA-100 in duplicate using the COL/EPI test cartridge. Patients in the No CAD group were younger, more often female, and less often diabetic, hypertensive, and lipidemic than CAD or Repeat CAD patients. Results: A short (〈 142 s) closure time (CT) in at least one of the duplicate PFA-100 COL/EPI cartridges occurred more frequently in CAD and Repeat CAD patients compared to No CAD patients (p 〈 0.05, Table). Conclusions: Failure of ASA to inhibit platelet function, as measured by the PFA-100, is associated with CAD, even in patients without prior history of CAD. The PFA-100, a shear-dependent system, may therefore be a useful test to assess the presence of ASA resistance. This test, unlike some tests for aspirin resistance, does not include ADP and therefore can be used in patients receiving thienopyridines (a frequent co-medication with aspirin). No CAD CAD Repeat CAD *p

Description: Eltrombopag, an orally-administered small-molecule agonist of the thrombopoietin receptor (c-Mpl), is under investigation as a treatment for immune thrombocytopenic purpura (ITP). Studies have indicated that eltrombopag does not ‘prime’ platelets for activation in vitro, and eltrombopag administration to healthy volunteers does not increase platelet surface P-selectin or activated integrin αIIbβ3 (Jenkins J. Blood 2007). However, the effects of eltrombopag on platelet function in thrombocytopenic patients in vivo, either by direct binding to c-Mpl receptors on platelets or indirectly by altering the dynamics of platelet production and causing an influx of young, large platelets, is unknown. Whole blood flow cytometry, unlike other assays of platelet function, enables measurement of platelet function in the setting of marked thrombocytopenia (Michelson. Platelets, Elsevier, 2007). As a substudy of larger treatment studies, 17 adult patients with chronic ITP received eltrombopag at a starting dose of 50 mg daily, with the possibility of an increase to 75 mg daily after 3 weeks. Blood samples were drawn pre-treatment, and after 7 and 28 days of therapy. Platelet count, mean platelet volume (MPV), and the immature platelet fraction (IPF, or reticulated platelet count) were measured using a Sysmex XE-2100. Platelet surface P-selectin and activated integrin αIIbβ3 (reported by monoclonal antibody PAC1) were measured by whole blood flow cytometry in the presence and absence of 0.5 μM ADP, 20 μM ADP, 1.5 μM thrombin receptor activating peptide (TRAP), or 20 μM TRAP. Bleeding was quantified by a comprehensive scale that allocates grades of 0 (no), 1 (minor) or 2 (marked) bleeding at 10 anatomical sites according to physical examination and/or history (Page, L.K. Br J Haematol 2007). Eleven of the 17 patients responded to eltrombopag with a rise in platelet count of 〉30 x 109/L. The IPF increased in responders but not non-responders (table 1). Response to eltrombopag was not predicted by pretreatment MPV or IPF. The ITP bleeding score decreased in responders over the study period in parallel with the increases in platelet count (table 1). As determined by platelet surface P-selectin and activated integrin αIIbβ3, eltrombopag did not result in platelet activation or augment ADP- or TRAP-induced platelet activation (table 2). In summary, eltrombopag increases the platelet count and reduces bleeding in responding adult patients with chronic ITP through the release of new platelets into the circulation. While bleeding is reduced in responders, eltrombopag does not result in platelet activation or augmentation of platelet activation by ADP or TRAP. This suggests that the newer platelets released by eltrombopag stimulation are not hyper-functional (or are only transiently so prior to day 7). Table 1 IPF (maximum absolute change, mean ± SEM x 109/L) Number in whom bleeding decreased Responders 57.0 ± 22.4 8/11 Non-responders 3.3 ± 1.5 1/6 Table 2 Study Day 0 7 28 MFI, mean fluorescence intensity, *P

Description: Introduction Nitric oxide (NO) released by endothelial cells interacts with platelets in which it stimulates soluble guanylate cyclase (sGC), thereby increasing platelet cyclic guanosine monophosphate (cGMP) and inhibiting platelet activation. Stimulation of sGC in other cells has been suggested as an attractive target for intervention in a range of diseases including pulmonary arterial hypertension, heart failure, and diabetes mellitus. Riociguat, the first FDA-approved sGC stimulator, potently increases platelet cGMP and inhibits platelet aggregation in washed platelets. Because riociguat binds to plasma proteins, higher concentrations of riociguat are required to inhibit platelet function in whole blood. However the potential synergistic inhibition of platelet function by riociguat and NO has not been well studied. Goal To investigate the possible synergistic effects of riociguat and NO on platelet inhibition and to determine the effects of protein binding. Methods Platelet-rich plasma (PRP) was prepared from citrate (3.2%) anticoagulated whole blood collected from healthy donors following informed consent. Riociguat (10 mM) in DMSO and DETA-NONOate 10 mM (an NO donor) in 10 mM NaOH were stored at -80°C until use. PRP was diluted 10-fold in either HEPES-Tyrode's buffer or platelet poor plasma (PPP), then incubated with vehicle or riociguat 1, 10, or 100 µM, alone or in combination with DETA-NONOate 16, 31, or 250 µM for 30 minutes, then analyzed by flow cytometry. Platelet surface activated GPIIb-IIIa (detected by monoclonal antibody PAC1) and platelet surface P-selectin were measured with and without activation by ADP 5 µM or thrombin receptor activating peptide (TRAP) 5 µM. For light transmission platelet aggregation (LTA) and 96-well platelet aggregation, PRP was used without dilution. Results For PRP diluted in buffer, riociguat and DETA-NONOate each produced concentration-dependent inhibition of ADP- and TRAP-stimulated platelet activation, as reported by platelet surface activated GPIIb-IIIa (Figure A) and P-selectin, and a synergistic inhibitory effect was observed when riociguat and DETA-NONOate were combined (for platelet surface activated GPIIb-IIIa, 40% inhibition with 1 µM riociguat alone; 30% inhibition with 31 µM DETA-NONOate alone; 90% inhibition with 1 µM riociguat and 31 µM DETA-NONOate combined). In contrast, when PRP was diluted in PPP, the concentrations of riociguat alone and DETA-NONOate alone needed to inhibit activation were dramatically increased and the combination of 1 µM riociguat and 31 µM DETA-NONOate produced less than 10% inhibition of platelet surface activated GPIIb-IIIa (Figure B). Synergistic inhibition in plasma was observed when DETA-NONOate was increased to 250 µM. Based on these results, a sub-threshold concentration of DETA-NONOate was chosen for investigation of the effects of riociguat on platelet aggregation. Using ADP 5 µM, TRAP, 2 µM, or collagen 2 µg/mL, riociguat alone at 10 µM (Figure C) and DETA-NONOate alone at 31 µM showed no inhibition of platelet aggregation. However, in the presence of 31 µM DETA-NONOate, riociguat showed a concentration-dependent inhibition of aggregation by each agonist (Figure C). Conclusions Platelets exposed to riociguat in combination with sub-threshold concentrations of NO, such as may occur in microvessels adjacent to the endothelial layer, are inhibited from undergoing platelet activation and aggregation. The presence of plasma proteins blunts the effects of riociguat and has even larger effects on the NO donor, DETA-NONOate. Taken together, these data suggest that NO potently synergizes with riociguat to inhibit platelet activation and aggregation, but in vivo this effect likely only occurs immediately adjacent to endothelial cells where NO concentrations are highest. Figure. Figure. Disclosures Michelson: Alnylam, Instrumentation Laboratory, Surface Oncology: Consultancy; AstraZeneca, Chiesi, Dova, Janssen, LightIntegra, Megakaryon, Remora: Other: Scientific Advisory Board; Baxalta, Cellular Preservation Technologies, Ionis, Ironwood, Medtronic, Megakaryon, Pfizer, Sysmex: Research Funding. Frelinger:Surface Oncology: Consultancy; Cellular Preservation Technologies, Ironwood, Ionis, Medtronic, Megakaryon, Pfizer and Sysmex: Research Funding.

Description: Key Points ITP patients differ in their tendency to bleed despite similarly low platelet counts, thereby confounding treatment decisions. Platelet function tests, independent of platelet count, are associated with bleeding severity in ITP patients.

Description: Key Points This breakthrough involves the role of the aryl hydrocarbon receptor in the expansion and specification of hematopoietic progenitor cells. This work sets a precedent for the use of an in vitro platform for the clinically relevant production of blood products.

Description: Abstract 1429 Introduction: Wiskott-Aldrich syndrome (WAS) is a complex disease in which patients suffer from both bleeding and immunodeficiency. Thrombocytopenia is severe, platelets are small and may be dysfunctional; intracranial hemorrhage occurs in 20% of patients. In patients with the full WAS phenotype, early use of human stem cell transplantation (HSCT) corrects the immune deficiency and thrombocytopenia although platelet transfusions are often required. Bleeding is the main complication of the X-linked thrombocytopenia (XLT) form, whose management may include HSCT, splenectomy, or supportive care; IVIG has limited benefit. Platelet function in WAS/XLT patients with low platelet counts has not been reported because of the inability to accurately perform standard assays in severely thrombocytopenic patients. The present study evaluated platelet function and thrombopoietin (TPO) mimetic (romiplostim or eltrombopag) treatment of thrombocytopenia in patients with WAS/XLT. Methods: Flow cytometry, which enables evaluation of platelet function despite thrombocytopenia, was used to study platelets in 8 WAS/XLT patients and age-matched normal controls. Platelet function was measured by: surface expression of P-selectin and activated GPIIb-IIIa (reported by PAC1) in whole blood following stimulation with low and high dose ADP and thrombin receptor activating peptide (TRAP); annexin V binding, a marker of platelet surface expression of the procoagulant phospholipid phosphatidylserine, in platelet-rich plasma (normalized to 30,000 platelets/μL) following stimulation with convulxin (a specific agonist of the platelet collagen receptor GPVI). The effects of romiplostim (10 μg/kg/wk SQ) or eltrombopag (50-75 mg/day PO) on platelet counts and bleeding were evaluated in 4 patients (3 WAS, 1 XLT). Results: Platelets from WAS/XLT patients showed reduced TRAP-induced platelet surface P-selectin and activated GPIIb-IIIa (p