A Comparative Proteomic Analysis of Platelets from Healthy Human Patients and Chronic ITP Patients Receiving AMG 531, a Thrombopoiesis-Stimulating Protein.

AMG 531 is a novel thrombopoiesis-stimulating “peptibody” consisting of an Fc carrier domain linked to 4 identical peptides that bind to and activate Mpl, the human thrombopoietin receptor. It has proven efficacy in raising platelet counts of patients with chronic ITP, and is being evaluated in chemotherapy-induced thrombocytopenia and myelodysplastic syndrome. While platelets from healthy subjects receiving AMG 531 reportedly behave normally in platelet aggregation studies, the effects of this medication on protein expression profiles are not known. The platelets from 12 healthy patients without known platelet defects and 9 chronic ITP patients receiving AMG 531 were isolated and their proteins extracted. Proteins with isoelectric points between pH 3 and 7 were separated by two-dimensional gel electrophoresis and stained with Sypro protein detection reagent. These gels were scanned and compared using Progenesis SameSpots software. In this pH range, there were approximately 5000 unique spots detected, representing individual proteins, isoforms of proteins or cleavage products. On multivariate analysis, approximately 11% of these proteins were expressed at levels significantly different between healthy patients without a known platelet disorder and chronic ITP patients receiving AMG 531. Principal component analysis substantiates these differences and a protein dendrogram has been created. The most significant protein expression differences are identified by tandem mass spectrometry. It is not known if these differences in protein expression represent younger platelets, stimulation of protein synthesis by AMG 531, or altered platelet protein expression as a reflection of the ITP. There is no known clinical correlate of these protein changes.