ALBERT

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: 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

Description: Abstract 5147 Platelet activation/aggregation in sickle cell disease (SCD) may promote tissue ischemia, suggesting antiplatelet therapy may be useful. However, assessing platelet function and the effect of antiplatelet therapy in blood from SCD patients may be confounded by hemolysis with release of ADP. Here we evaluate levels of platelet activation markers in SCD adolescents vs. normal controls and compare, by multiple methods, the effect of in vitro blockade of the platelet ADP receptor P2Y12 by prasugrel's active metabolite, R-138727. Platelet activation markers in blood from SCD adolescents (n=15) and healthy adults (n=10), and the effect of R-138727 (0. 1 – 10 μM) added in vitro, were evaluated with and without ADP stimulation. Circulating levels of platelet-monocyte and platelet-neutrophil aggregates were significantly higher (p

Description: Cystic fibrosis (CF) is caused by a mutation of the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). We examined platelet function in CF patients because lung inflammation is part of this disease and platelets contribute to inflammation. CF patients had increased circulating leukocyte-platelet aggregates and increased platelet responsiveness to agonists compared with healthy controls. CF plasma caused activation of normal and CF platelets; however, activation was greater in CF platelets. Furthermore, washed CF platelets also showed increased reactivity to agonists. CF platelet hyperreactivity was incompletely inhibited by prostaglandin E1 (PGE1). As demonstrated by Western blotting and reverse-transcriptase-polymerase chain reaction (RT-PCR), there was neither CFTR nor CFTR-specific mRNA in normal platelets. There were abnormalities in the fatty acid composition of membrane fractions of CF platelets. In summary, CF patients have an increase in circulating activated platelets and platelet reactivity, as determined by monocyte-platelet aggregation, neutrophil-platelet aggregation, and platelet surface P-selectin. This increased platelet activation in CF is the result of both a plasma factor(s) and an intrinsic platelet mechanism via cyclic adenosine monophosphate (cAMP)/adenylate cyclase, but not via platelet CFTR. Our findings may account, at least in part, for the beneficial effects of ibuprofen in CF. (Blood. 2005;105:4635-4641)

Description: Because of methodologic problems, platelet function in thrombocytopenic patients has never been adequately studied, and therefore individual differences in bleeding risk are poorly understood. Whole blood flow cytometry analyzes the function of individual platelets, thereby enabling assessment of platelet function in severe thrombocytopenia (Michelson, A. Platelets, 2nd ed, Elsevier, 2007). In this study, platelet function was compared in 31 ITP patients, 14 CIT patients, and 16 healthy controls. Clinical bleeding was related to platelet function testing. The immature platelet fraction (IPF, or reticulated platelets) was measured in 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 μΜ TRAP, or 20 μM TRAP. Bleeding was quantified by a comprehensive score that allocates grades of 0 (no), 1 (minor) or 2 (marked) bleeding at 10 anatomic sites (Page, L.K. Br J Haematol 2007). Mean platelet volume (MPV) and IPF were higher in ITP than CIT, reflecting, as expected, a higher rate of platelet production (Table 1). Platelet surface P-selectin without added agonist (i.e. circulating activated platelets) was significantly higher in both ITP and CIT patients than in controls (Table 2). However, upregulation of platelet surface P-selectin and activated αIIbβ3 in response to ADP and TRAP (platelet ‘activatability’) was reduced in CIT patients compared with ITP patients and controls (Table 2). Stratification of bleeding scores by platelet count showed that CIT patients had more GI, urinary, and pulmonary bleeding at platelet counts