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/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.

Fig. 1.

Differential proteome analysis of normal donor and MDS platelets. Representative two-dimensional gel image of platelet lysates (pH ranges 4–7 and 6–9) showing the 120 protein spots with different concentrations between the seven MDS patients and seven normal donors analyzed. Red circles represent the 101 spots with lower levels in MDS platelets, and blue circles represent the 19 spots with higher levels in MDS platelets. The serial numbers are identical to the numbers given in Table I, showing the position of each of the identified proteins. Letters following numbers are used to distinguish between different spots representing the same protein.

Fig. 2.

Western blot of platelet lysates from MDS patients and healthy donors. The pooled platelet lysates from seven MDS patients and seven healthy donors show the decreased levels of Talin-1, Vinculin, Filamin-A, and Myosin-9 in MDS platelets.

Fig. 3.

Ingenuity network and pathway analysis. A, ingenuity network analysis using all identified proteins from our two-dimensional difference gel electrophoresis analysis. B, ingenuity pathway analysis of integrin signaling. Both show the up-regulated proteins in the MDS platelet in red and the down-regulated in green.

Fig. 4.

MDS platelet surface receptor characterization and primary activation. A, flow cytometry analysis of platelets from MDS patients and normal donors shows no difference in surface levels of integrin α_IIbβ₃ (GPIIb/IIIa), ADP receptors P2Y₁ and P2Y₁₂, as well as collagen receptors GPVI and α₂β₁ (VLA-α2) and von Willebrand factor receptor GPIb (n = 10). B, calcium mobilization from internal stores over time upon platelet activation with different agonists shows no difference between MDS and healthy donor platelets (n = 10). C, granule release upon platelet activation shows no difference between MDS and normal platelets (n = 11).

Fig. 5.

FRET analysis of talin-1 co-localization to the integrin β₃. A, bi-parametric analysis of emissions at 575 nm (PE, abscissa) and 675 nm (APC, ordinate) after excitation at 488 nm of MDS and healthy platelets labeled with anti-integrin α_IIbβ₃ conjugated to PE (upper panel) and in combination with anti-talin-1 conjugated to APC (lower panel) showing the reduced emission at 675 nm in MDS. B, box plot showing the significantly reduced MFI at 675 nm after excitation with 488 nm in MDS platelets compared with normal donors (n = 5, p < 0.05).

Fig. 6.

Flow cytometry analysis of MDS platelet activation. MDS platelets show a significant decrease in their PAC-1 MFI following TRAP and phorbol 12-myristate 13-acetate stimulation resembling insufficient integrin α_IIbβ₃ activation (n = 8, p < 0.05 and 0.01). Mn²⁺-stimulated platelets from MDS and normal donors show no differences with regard to PAC-1 binding (n = 3).

Fig. 7.

Defective spreading capacity of MDS platelets. A, differential interference contrast microscopy images of one representative experiment out of four showing that thrombin-stimulated MDS platelets fail to completely spread over a continuous time period of 30 min forming only occasional filopodia/lamellipodia. Images were taken using a Zeiss LSM 510 META microscope in combination with a Plan-Neofluar 40×/1.3 oil differential interference contrast objective and processed by Zeiss LSM Image Browser Software Version 4.2.0.121. Scale bar, 5 μm. B, quantification of spread platelets in normal donors and MDS patients (n = 4). For each patient and normal donor, 100 platelets were counted after 30 min, and the bar charts represent the proportion of fully spread platelets showing a significantly lower proportion in MDS (p < 0.001).

Fig. 8.

Integrin-dependent platelet aggregation defect in MDS. A, compared with normal donor platelets, MDS platelets (n = 58) showed a significantly reduced aggregation in response to collagen and arachidonic acid (p < 0.001 and 0.01). Aggregation following ristocetin and ADP stimulation was also reduced (p < 0.01 and 0.05). B, scatter plot of platelet count versus aggregation capacity of 58 MDS patients showing that in patients with lower platelet counts (<157,000/μl) defective aggregation in response to collagen and arachidonic acid is more common (p < 0.001).