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: 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: Introduction: With an estimated 15 million patients and no drug that addresses its etiology, sickle cell disease (SCD) remains an area of unmet need. Vaso-occlusive pain crisis (VOPC), the hallmark of SCD, is initiated by sickle RBCs (sRBCs) recruiting leukocytes and platelets to potentiate vessel occlusion. ADP released by sRBCs is a potent activator of platelets, and sickle cell patients are known to have activated platelets in circulation both at steady state and during VOPC. However, the mechanism underlying platelet dysfunction in SCD is not fully understood. Platelet activation mediated by the protease activated receptors (PAR1 and PAR4 in humans, PAR3 and PAR4 in mice), triggers PLC-β activation resulting in calcium mobilization. The increased calcium flux leads to activation of GPIIbIIIa/aIIbb3, GP1b, and P-selectin involved in platelet aggregation, adhesion, and rolling. Prior evidence has established a role of the calcium-activated cysteine protease, calpain-1 in platelet activation. Washed platelets from calpain-1 knockout C57BL/6 mice demonstrated impaired platelet aggregation. However, due to the critical contribution of sRBCs to platelet dysfunction in SCD, whole blood (impedance) aggregometry represents a physiological assessment of platelet aggregation. Methods: Townes SCD mice (SS) were backcrossed with calpain-1 knockout (CKO) mice to generate SCD mice lacking calpain-1 (SSCKO). Humanized mice (AA) were used as controls. Using flow cytometry, we evaluated in vivo platelet activation following stimulation with ADP and Thrombin GPRP. Platelet counts were obtained via ADVIA 120 and flow cytometry. For platelet aggregation, 500 μL of blood was harvested from the vena cava of AA, SS, SSCKO, and CKO mice. Whole blood aggregation in response to PAR4 stimulation was assessed using the Roche Multiplate Analyzer. A separate group of mice were challenged with hypoxia/reoxygenation (H/R) treatment (3 hours of 10.5% O2, followed by 4 hours of 21% O2) prior to platelet aggregation testing. SCD mice are characterized by tissue infarcts suggestive of thrombus formation. To examine whether H/R treatment induces formation of fibrin thrombi, we harvested brain, lungs, heart, kidneys, liver, and spleen following blood collection, and performed histology. Results: Compared to AA, SS and SSCKO mice are thrombocytopenic. Similar to Berkeley, Townes mouse platelets are activated in vivo, demonstrated by activated GPIIbIIIa on circulating platelets. At steady state, PAR4 agonist-induced platelet aggregation is similar in AA and SS mice (64 U v. 53 U, n = 6-10/group, p = 0.3). As depicted in Fig 1., SSCKO mice show significantly reduced platelet aggregation compared to SS mice (13 U v 53 U, p

Description: Diabetes mellitus (DM) patients have a 2–8 fold increased risk for cardiovascular disease (CVD) and suffer from microvascular (nephropathy, retinopathy) and macrovascular (peripheral arterial disease) complications. We demonstrated previously in normal platelets that insulin signals through Insulin Receptor Substrate-1 (IRS-1) and inhibits Ca2+ mobilization by interfering with the Gi alpha-mediated suppression of cAMP, a platelet inhibitor (Ferreira et al, JBC 2004). Platelets from type 2 DM patients have become insulin- resistant and their platelets aggregate better than controls possibly as a result of up-regulation of P2Y12 mediated suppression of cAMP (Ferreira et al, ATVB 2006). This hyper-aggregability might be a cause for the increased CVD risk. In the present project we searched for the cause of insulin resistance in type 2 DM platelets. Since most of the properties of platelets are determined by the megakaryocyte and obese subjects are prone to develop insulin resistance, we investigated whether adipokines change the control of Ca2+ mobilization by insulin in the megakaryocyte. To suspensions of megakaryocytic CHRF-288-11 cells incubated with adipokines and plasma from obese individuals or controls, we added insulin and measured inhibition of thrombin-induced Ca2+ mobilization as indicator of insulin sensitivity, phosphorylation of IRS-1 [Ser307] and protein kinase Balpha [Ser473] as indicators of insulin signaling, and compared results with the expression of Suppressor Of Cytokine Signaling (SOCS) proteins which is under control of adipokines. Insulin inhibited Ca2+ mobilization by 20 ± 7% at 1 nM (p

Description: Background. Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder that results in thrombocytopenia and the risk of bleeding. Severe bleeding, while uncommon, is partially associated with the degree of thrombocytopenia. However it remains unclear why some thrombocytopenic patients with ITP have significant bleeding while others have minimal or no bleeding manifestations. We hypothesized that differences in platelet function between ITP patients contribute to the variation in bleeding tendency and these differences can be used to identify children at the highest risk for bleeding. We recently reported (Frelinger et al. Blood 2015;126:873-879) that platelet tests related to platelet age (immature platelet fraction [IPF], forward light scatter [FSC]) and activation through the PAR1 thrombin receptor (TRAP-stimulated P-selectin, activated GPIIb-IIIa, and GPIba) are associated with concurrent bleeding severity in ITP, independent of the platelet count. Aims. 1) Determine the association of platelet function tests with subsequent bleeding severity. 2) Determine the consistency of the platelet function phenotype over time. Methods. At two study visits separated by at least 1 month, bleeding severity (graded by the Buchanan and Adix Score) and platelet function tests were evaluated in a single center cross-sectional study of patients ≥6 months of age with a diagnosis of ITP or Evans syndrome. Platelet function was assessed by whole blood flow cytometric analysis of platelet surface P-selectin, activated GPIIb-IIIa (measured by monoclonal antibody PAC1), and GPIbα with and without in vitro agonist (ADP or thrombin receptor activating peptide [TRAP]) stimulation. Complete blood cell counts, including IPF, were obtained in a Sysmex XN-1000. Results. Fifteen patients (9.5 ± 5.2 [mean ± SD] years of age, 4 male) were evaluated with a median interval between visits of 10.1 months (interquartile range 2.3 - 23.9 months). Bleeding severity was lower at Visit 2 compared to Visit 1 (Visit 1: Grade 0, n=4, Grade 1, n=4, Grade 2, n=6, Grade 3, n=1 vs. Visit 2: Grade 0, n=7, Grade 1, n=6, Grade 2, n=1, Grade 3, n=1, p = 0.0107). At Visit 2 mean platelet count was higher and IPF was lower than at Visit 1 (111 vs. 69 x 109 platelets/L, p = 0.06 and 10.7 vs. 14.3% IPF, p = 0.04). Nevertheless, platelet count and IPF at Visit 1 were both strongly correlated with platelet count and IPF, respectively, at Visit 2. At each visit, % P-selectin-positive platelets with 20 µM ADP, % PAC1-positive with 20 µM ADP, and GPIba mean fluorescence with 1.5 and 20 µM TRAP, were associated by univariate analysis with the concurrent bleeding score (Figure). Platelet function in patients with ITP was consistent over time as demonstrated by: a) significant correlations between platelet count, IPF, and circulating or agonist-stimulated platelet surface P-selectin, activated GPIIb-IIIa, and GPIba at Visit 1 vs. Visit 2; b) significant associations between platelet markers at each visit with bleeding scores at each visit; and c) a significant association of platelet markers at Visit 1 with bleeding scores at Visit 2 (Figure). Conclusions. Platelet function tests identify a platelet phenotype in children with ITP that is consistent over time and is associated with bleeding severity at both concurrent and subsequent visits. These results suggest the platelet phenotype contributes to the bleeding risk in children with ITP and supports further evaluation of platelet function testing to help guide patient management. Figure Univariate association of platelet tests with bleeding score at concurrent and subsequent visits. Tests significantly associated with bleeding score in all comparisons are highlighted. Figure. Univariate association of platelet tests with bleeding score at concurrent and subsequent visits. Tests significantly associated with bleeding score in all comparisons are highlighted. Disclosures Frelinger: Sysmex: Research Funding. Grace:Agios Pharmaceuticals: Other: Scientific Advisor, Research Funding. Michelson:Sysmex: Research Funding.

Description: Background Immune thrombocytopenia (ITP) patients with similarly low platelet counts differ in their tendency to bleed. Aim To determine if differences in platelet function in ITP patients with similarly low platelet counts partly account for the variation in bleeding tendency. Methods The relationship between bleeding scores and platelet function markers was investigated in a single center cross-sectional study of pediatric patients with ITP. Following informed consent, blood was collected from ITP patients and bleeding was graded using the Buchanan and Adix Score (J Pediatr 2002) at routine clinic visits or while admitted to the hospital. Bleeding scores were obtained by one of three hematologists blinded to platelet function results, and investigators performing platelet function tests were blinded to clinical results. Platelet function was assessed by whole blood flow cytometric measurement of unstimulated, ADP- or TRAP-stimulated platelet surface activated GPIIb-IIIa (as measured by PAC1 binding), P-selectin, and GPIb and by unstimulated, convulxin-, or ADP plus TRAP-stimulated platelet surface phosphatidylserine expression (as determined by annexin V binding). Platelet count, immature platelet fraction (IPF) and mean platelet volume (MPV) were determined by a Sysmex XE-2100, and platelet forward angle light scatter (FSC) was measured by flow cytometry. Results Platelet function and bleeding scores were evaluated in 34 consecutive consenting pediatric ITP patients (16 female, 18 male, age 9.7 ± 5.7 years [mean ± SD]). ITP was newly diagnosed (〈 3 months) in 10 patients, persistent (3 -- 12 months) in 7 patients, and chronic (〉12 months) in 17 patients. Platelet count at the time of the blood draw was 47 ± 55 x 109/L. The median bleeding score on day of blood draw was 1 (range 0 to 4). By univariate analysis, higher IPF, and lower platelet count were significantly associated with a higher bleeding score (odds ratio [OR] 〉1, p1, p

Description: Key Points Platelet function in WAS/XLT, measured by agonist-induced surface-activated GPIIb-IIIa and P-selectin, is proportional to platelet size. Eltrombopag increased platelet counts, but did not improve platelet activation, in most WAS/XLT patients.

Description: Patients with Glanzmann thrombasthenia or Leukocyte Adhesion Deficiency-III syndrome (LAD-III or LAD-1/variant) present with increased bleeding tendency because of the lack or dysfunction of the fibrinogen receptor GPIIb/IIIa (integrin αIIbβ3), respectively. Although the bleeding disorder is more severe in LAD-III patients, classic aggregometry or perfusion of Glanzmann or LAD-III platelets over collagen-coated slides under physiologic shear rate does not discriminate between these 2 conditions. However, in a novel flow cytometry-based aggregation assay, Glanzmann platelets were still capable of forming small aggregates upon collagen stimulation, whereas LAD-III platelets were not. These aggregates required functional GPIa/IIa (integrin α2β1) instead of integrin αIIbβ3, thus explaining the clinically more severe bleeding manifestations in LAD-III patients, in which all platelet integrins are functionally defective. These findings provide genetic evidence for the differential requirements of platelet integrins in thrombus formation and demonstrate that correct integrin function assessment can be achieved with a combination of diagnostic methods.