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Abstract
Bleeding complications are a significant clinical problem in patients with myelodysplastic syndromes even at sufficient platelet counts (>50,000/μl). However, the underlying pathology of this hemorrhagic diathesis is still unknown. Here, we analyzed the platelet proteome of patients with myelodysplastic syndromes by quantitative two-dimensional difference gel electrophoresis followed by mass spectrometric protein identification. Proteins identified with lower concentrations, such as Talin-1, Vinculin, Myosin-9, Filmain-A, and Actin play critical roles in integrin αIIbβ3 signaling and thus platelet aggregation. Despite normal agonist receptor expression, calcium flux, and granule release upon activation, the activation capacity of integrin αIIbβ3 was diminished in myelodysplastic syndrome platelets. Förster resonance energy transfer analysis showed a reduced co-localization of Talin-1 to the integrin's β3-subunit, which is required for receptor activation and fibrinogen binding. In addition, platelet spreading on immobilized fibrinogen was incomplete, and platelet aggregation assays confirmed a general defect in integrin-dependent platelet aggregation in patients with myelodysplastic syndromes. Our data provide novel aspects on the molecular pathology of impaired platelet function in myelodysplastic syndromes and suggest a mechanism of defective integrin αIIbβ3 signaling that may contribute to the hemorrhagic diathesis observed in these patients.
Highlights
From the Departments of §Hematology, Oncology, and Clinical Immunology and ¶Clinical Biochemistry and Pathobiochemistry, Leibniz Center for Diabetes Research, and ʈInstitute for Transplantation Diagnostics and Cell Therapeutics, Heinrich-Heine-University, 40225 Dusseldorf, Germany and the **Department of Cell Biology and Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, New York 10461
Despite some evidence that a functional platelet defect may exist in Myelodysplastic syndromes (MDS), no data are yet available to explain the hemorrhagic diathesis in those patients who present with sufficient platelet counts
The decreased expression of Talin-1, Myosin-9, Filamin-A, and Vinculin was confirmed by Western blot showing a 23% decrease of Talin-1 amount in MDS platelets compared with healthy donor platelets as well as 42% of Vinculin, 17% of Filamin-A, and 63% of Myosin-9, respectively, -tubulin levels were not altered (Fig. 2)
Summary
Patient Characteristics—The 66 MDS patients included into this study had a median age of 66 years and a median platelet count of 100,000/l and did not receive platelet transfusion or any medication possibly interfering with platelet function. Changed protein spots were determined by the following three-step procedure: 1) the spot was matched across at least 10 of 14 gels; 2) its standardized average spot volume ratio exceeded 1.5 in at least five of the seven patients compared with the normal donor group, and 3) this change in spot volume was statistically significant (p Ͻ 0.05). Forster Resonance Energy Transfer (FRET)—Washed platelets from five MDS patients and normal donors were incubated with 1 unit/ml thrombin and simultaneously stained for 20 min at RT in the dark with anti-CD41 (Acris Antibodies) and anti-talin-1 (LSBio) conjugated to phycoerythrin (PE) and allophycocyanin (APC) using Lynx Rapid Conjugation kits (AbD Serotec). Whole Blood Flow Cytometry—Within 30 min after blood sampling, l of citrate-anticoagulated whole blood from MDS patients and normal donors was diluted in PBS, incubated with agonists, simultaneously stained with antibodies for 20 min at RT in the dark, and subsequently analyzed using a FACSCalibur (BD Biosciences). Established methods for correction lowered the number of false-positive discoveries at the expense of increasing false-negative discoveries. 2 automatic interaction detection was used for modeling the dependence of the aggregation defect on multiple clinical parameters (all SPSS 19, IBM)
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