Abstract 270: Immune vs. Thrombotic Stimulation of Platelets Differentially Regulates Intracellular Protein Associations and Protein Release: a Proteomics Analysis
In addition to hemostasis, platelets mediate inflammation and the clearance of bacteria from the bloodstream. As with platelet-platelet interactions, platelet-bacteria interactions involve release of granular content and cytoskeletal rearrangements in the platelet. Recently, we identified Toll-like receptors (TLRs) on the platelet surface. TLR2 in particular recognizes the bacterial lipopeptide Pam3CSK4 and leads to platelet aggregation. However, the differential regulation of intracellular proteins and granule release in immune vs. thrombotic responses is not well understood in platelets. We studied platelet Factor XIIIA (FXIIIA) as a unique candidate protein, as the released form regulates clot stabilization and the cytosolic form crosslinks cytoskeletal proteins upon platelet activation. Our hypothesis, that the activation of platelets by thrombotic or immune pathways leads to differential regulation of protein interactions and granule release, was tested using a proteomics approach. We immunoprecipitated FXIIIA from resting and activated platelets to study FXIIIA-associated proteins by gel electrophoresis in one or two dimensions (2D-GE), followed by mass spectrometric identification of protein spots. Results were confirmed by Western blot and confocal microscopy. Proteins released after stimulation with Pam3CSK4 or thrombin were analyzed by 2D-GE and Western blotting. We identified 15 proteins that associate with cytosolic or granular FXIIIA in resting platelets. Several novel protein interactions were found such as talin, GAPDH, gelsolin, tubulinβ. Specific protein associations were differentially altered by immune or thrombotic stimulation including gelsolin (20% decrease vs. no change) and GAPDH (80% vs. 30% decrease). Interestingly, integrin α IIb showed specific association with FXIIIA after thrombin- but not Pam3CSK4-activation. In addition, about 80 released proteins from Pam3CSK4- vs. thrombin-stimulated platelets were differentially displayed, including FXIIIA. In summary, immune or thrombotic stimulation of platelets leads to differential protein associations and protein release. These findings highlight the differences in the platelet’s inflammatory vs. thrombotic response.