Platelets Derived From Patients with Myelodysplastic Syndromes (MDS) Show Defective Platelet Aggregation and Integrin aIIbβ3 Receptor (GPIIb/IIIa) Signaling Caused by a Reduced Expression of Proteins Required for Inside-out Signaling.

Abstract 598

Thrombocytopenia is prevalent in up to 65% of patients with myelodysplastic syndrome (MDS) at the time of diagnosis and thrombocytopenic hemorrhage is a significant clinical problem that is often complicated by platelet aggregation defects. Little is known about the pathophysiology of this insufficient platelet function. Here, we delineate a reduced expression of critical platelet aggregation-related proteins by analyzing the platelet proteome of 7 patients with MDS and 7 normal donors. Patients' median platelet count was 60 × 10E9/L (range 37–109 × 109/L) and none of the patients examined had received prior anticoagulant treatment, chemotherapy or platelet transfusions. Differential 2-dimensional in-gel electrophoresis coupled with a time-of-flight Ultraflex-Tof/Tof mass spectrometer enabled the discovery of 120 differential protein spots. Among these, we identified a total of 35 proteins including 26 proteins that are integral part of the integrin aIIbβ3 receptor (GPIIb/IIIa, Fibrinogen receptor) signaling such as Talin-1 and Vinculin. In resting platelets the integrin aIIbβ3 receptor exhibits a low-affinity (inactive) state which is shifted to a high-affinity (active) state following inside-out activation. Talin-1 expression has been shown to be essential for this inside-out activation of the integrin aIIbβ3 receptor and consecutive platelet aggregation in an in-vivo model. We hypothesized that the reduced expression of Talin-1 and its co-factor Vinculin may inhibit activation of the integrin aIIbβ3 receptor and thereby contribute to the platelet aggregation defect seen in patients with MDS. Therefore, we performed further functional studies on integrin aIIbβ3 receptor activation and platelet spreading/aggregation with platelets derived from an independent cohort of 7 patients with MDS and 7 normal donors. In this new cohort, patients' median platelet count was 94 × 109/L (range 60–120 × 109/L) and again all patients had never received prior platelet transfusions or anti-coagulant treatment. When we looked at the surface expression of the integrin aIIbβ3 receptor on resting platelets by means of flow-cytometry, we did not detect any differences between platelets from patients with MDS and normal donors. Then, we activated platelets from normal donors and patients with MDS with 0.01U/μl and 0.001U/μl thrombin and measured binding of PAC-1, which is highly specific for detection of the active form of the integrin aIIbβ3 receptor. Here, we found a significantly lower shift from the inactive to the active form in platelets derived from patients with MDS dropping from 92.15% and 91.46% in normal donors to 41.97% and 48.45% (p = 0.01 and p = 0.006), respectively. We confirmed this suggested lack of adhesion and aggregation capacities in MDS platelets by confocal microscopy and single platelet imaging of washed platelets stimulated with 0.01U/μl thrombin which were adhered to immobilized fibrinogen. Consecutive platelet aggregation assays also revealed an insufficient response to stimuli like Collagen and ADP. Our findings provide for the first-time insight into the molecular pathology of defective platelet aggregation in MDS and suggest a mechanism of defective inside-out signaling caused by a reduced expression of proteins required for integrin aIIbβ3 receptor activation.