Platelet Proteome Analysis Reveals Integrin-dependent Aggregation Defects in Patients with Myelodysplastic Syndromes

Abstract

Bleeding complications are a significant clinical problem in patients with myelodysplastic syndromes even at sufficient platelet counts (>50,000/l). 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 ␣ IIb ␤ 3 signaling and thus platelet aggregation. Despite normal agonist receptor expression, calcium flux, and granule release upon activation, the activation capacity of integrin ␣ IIb ␤ 3 was diminished in myelodysplastic syndrome platelets. Fö rster resonance energy transfer analysis showed a reduced co-localization of Talin-1 to the integrin's ␤ 3 -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 ␣ IIb

What Can Proteomics Tell Us About Platelets?

“…In one study, proteins from resting and collagen receptor-stimulated platelets were incubated with cAMP/cGMP-coupled beads, and the pull-down eluates were analyzed by LC-MS/MS. 115 Owing to the enrichment provided by specific probes, activity-based protein profiling 116-121 B, These studies are mainly (40%) based on 2-dimensional gel electrophoresis (2-DE)/ difference gel electrophoresis (DIGE), 46,52,53,80,82,83,85,87,88,90,91,[93][94][95][96]98,102,106,110,112,113 whereas gelfree methods (23%) are still under-represented. 21,42,89,97,[114][115][116][117][118]121 Combination refers to studies using 2-DE/DIGE in combination with other approaches.…”

Proteomic profiling of platelet signalling

“…Comparison of the proteomes from myelodysplastic and control platelets by 2-DE/MALDI MS identified decreased levels of the structural proteins talin-1, vinculin, myosin-9, filamin-A and actin [71]. The co-localization of talin-1 to integrin β3 was impaired as was platelet spreading on immobilized fibrinogen and aggregation; providing a possible explanation for the hemorrhagic defects associated with this condition.…”

“…Filamin NO platelet activation 2-DE/MALDI [67,71] 14-3-3ζ NO platelet activation 2-DE/MALDI [67] PI3K-γ NO platelet activation 2-DE/MALDI [67] F-Actin capping protein ASA-resistance 2-DE/MALDI [64] HSP60 ASA-resistance 2-DE/MALDI [64] HSP71 ASA-resistance 2-DE/MALDI [64] SPARC ACS 2-DE/MALDI [68] CrkL STEMI 2-DE/MALDI [70] Src STEMI 2-DE/MALDI [70] Talin-1 MDS 2-DE/MALDI [71] Vinculin MDS 2-DE/MALDI [71] Myosin-9 MDS 2-DE/MALDI [71] Filamin-A MDS 2-DE/MALDI [71] Actin MDS 2-DE/MALDI Review their platelets display decreased spreading on fibrinogen-coated surfaces [47]. In order to elucidate the signaling pathways associated with PAR-1 activation, Garcia et al compared the proteomes of resting platelets and those stimulated with TRAP using 2-DE and LC-MS/MS [48].…”

Diagnostics of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML)

“…Molecular markers will soon be integrated with prognostic scoring systems, and likely become therapy-relevant and predictive in nature. More recent analytical methods, such as proteomics, are of great scientific interest, but not yet part of routine diagnostics [15][16][17]. Table 5 summarizes standard MDS diagnostics [18,19].…”