Exploring the interaction environment of blood cells : proteomic analysis of platelet releasate and platelet-monocyte interaction

Platelet-monocyte aggregates circulating in the blood are found to play an important role in cardiovascular disease, the most common cause of death in Canada, and are now an established early marker of acute events. Upon stimulation, monocytes transmigrate across the endothelial layer into the intima where they take up oxidized low density lipoproteins (LDL) and differentiate into macrophages, and are then incorporated into atherosclerotic plaques. Upon plaque rupture, which accounts for the majority of fatal cardiovascular incidents, platelets are exposed to a variety of agonists such as collagen and mildly oxidized LDL. We used dimethyl labeling quantitative proteomics approach to determine the relative abundance of platelet releasate (Rel) proteins from platelets activated with thrombin, collagen or Lysophosphatidic acid (LPA; the most potent platelet activator found in mildly oxidized LDL). Using the different agonists led to releasates with unique protein compositions. In addition, we analyzed the relative abundance of protein in releasate free of microparticles (Rel-MP) when using the different agonists. Flow cytometry and proteomics studies showed that thrombin, collagen or LPA activated platelets not only produce different number of platelet microparticles (MP), but that these MP have different proteome profiles. We studied the effects of combining agonists by activating platelets with thrombin plus collagen or a subthreshold concentration of collagen plus LPA. Through biologic functional studies we saw that the Rel, Rel-MP or MP from the platelets activated with the different agonists lead to different degree of THP-1 cell migration. We added the contents released from thrombin activated platelets to a human monocytic cell line THP-1, in order to find the proteins which are responsible for THP-1 cell stimulation. Based on the increased expression of proteins such as integrin β1, we found that, adding platelet releasate induces a pro-inflammatory state in THP-1 cells and prime them for transmigration. Therefore, we have taken considerable strides towards uncovering the effect of platelet activation on monocyte protein expression. The findings may aid in discovery of drug targets for prevention of inappropriate platelet activation and platelet-monocyte aggregate formation, in order to dampen the effects of these events in contributing to cardiovascular disease.Medicine, Faculty ofMedicine, Department ofExperimental Medicine, Division ofGraduat