Identification of Kinase-Dependent Signaling Pathways Regulated By Rap1b in Murine Platelets

Identifying signaling pathways regulated by Rap1b has been challenging using classical biochemical techniques. Using quantitative iTRAQ- and TiO2-based phosphoproteomics, we sought to identify protease-activated receptor 4 (PAR4) thrombin receptor-activated kinase cascades influenced by Rap1b in murine blood platelets. We identified a total of 1,783 phosphopeptides, and after screening for activation dependence, we found 7 phosphopeptides from 4 proteins that were >1.5-fold more abundant in WT compared to Rap1b-null mouse platelets and 5 phosphopeptides from 4 proteins were >1.5-fold more abundant in Rap1b-null. One protein that showed higher phosphorylation in activated WT platelets was Talin, which had 3 sites of increased phosphorylation: T430, S446, and S1201. The functions of these sites have yet to be determined, and the local sequences near these sites do not match any known kinase consensus sequences. Two of these sites, T430 and S446, are in close proximity to the Calpain cleavage site of Talin at Q432-Q433. We found Calpain cleavage of Talin was greater in PAR4-TRAP-activated Rap1b-null platelets compared to WT after 5 minutes of PAR4 stimulation. If phosphorylation inhibits Talin cleavage by Calpain, a possible explanation for the platelet defects seen in Rap1b-null platelets may be that cleavage of Talin reduces Talin clustering, and therefore integrin clustering, leading to reduced aggregation and reduced adhesion under shear. Other proteins identified include RhoGAP 18 (more abundant in WT) and ERK1/2 (more abundant in Rap1b-null). In conclusion, we have 1) used an unbiased proteomic approach to search for downstreams effectors of Rap1b during PAR4-mediated platelet activation, 2) identified 12 phosphopeptides from 8 unique proteins that had >1.5-fold difference in activated platelets from Rap1b-null mice versus WT mice, including Talin, RhoGAP18, and ERK1/2, and 3) identified a difference in Calpain-mediated Talin cleavage between activated plateles from Rap1b-null mice versus WT mice. These results suggest a signaling cascade from Rap1b to Talin, a known regulator of integrin activation. This work is supported by NIH Grant HL45000 and Blood Center Research Foundation.