This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Beside hemostasis, platelets are known to play a role in immune responses and inflammatory processes. Interaction with pathogens involves pathogen recognition, release of granule-content, and cytoskeletal rearrangements in the platelet. We have identified the expression of toll-like receptors (TLR) on the platelet surface, supporting a role for the platelet in innate immune responses. In addition, the TLR pathway links inflammation to atherosclerosis, as TLRs are expressed in atherosclerotic plaques and murine TLR2 stimulation causes enhanced atherosclerosis. Intracellular Factor XIIIA (FXIIIA) can crosslink platelet cytoskeletal proteins upon platelet activation. Relevant to the immune response, FXIIIA is required for cytoskeletal remodeling during monocyte phagocytosis, but its role in platelet-mediated immune processes is unknown. We have applied a proteomics approach to search for TLR2- and FXIIIA-associated proteins in resting platelets. After immunoprecipitation (IP) of TLR2 and FXIIIA from platelet lysates followed by gel separation, protein bands were excised, digested with trypsin and analysed by mass spectrometry. Western blot analysis was applied to confirm the mass spectrometric results. We repeatedly immunoprecipitated FXIIIA directly with TLR2-specific antibodies, whereas the IP of FXIIIA pulled down a truncated form of TLR2, as identified by Western blot, that may correspond to the soluble form of TLR2. By mass spectrometry, we identified FXIIIA and FXIIIA-associated proteins: thrombospondin, filamin and talin. Importantly, talin has not been previously reported as FXIII-substrate or associated protein. Thrombospondin, a known FXIII substrate, is stored in platelet granules. A strong thrombospondin-band after FXIIIA-IP and a weak band after TLR2-IP suggests the partial presence of FXIIIA and soluble TLR2 in granules. Interestingly, FXIIIA appears to be associated with its substrates in the resting platelet. In conclusion, our results indicate the presence of known and novel FXIIIA-associated proteins in platelets and suggests a role for TLR2 and FXIIIA in platelet-dependent immune responses. A manuscript reporting these results has been accepted for publication in Thrombosis and Hemostasis .
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