Lightning Talk & Poster 28th Annual Lorne Proteomics Symposium 2023

Defining the Structures and Interactions of the Human Platelet Secretome using Chemical Crosslinking (#23)

Michelle cielesh 1 , Freda H Passam 2 , Mark Larance 1
  1. Charles Perkins Centre, School of Medical Sciences, University of Sydney, Sydney, NSW, Australia
  2. Heart Research Institute, The University of Sydney, Sydney, NSW, Australia

Platelet activation induces the secretion of proteins that promote platelet aggregation and inflammation. However, the understanding of the regulation and function of these proteins, many of which are only expressed in platelets, has been hampered by a lack of tertiary structure information and limited knowledge of protein-protein interactions. Here, we detail the first chemical crosslinking analysis of the platelet secretome (releasate) using DSSO and nanoLC-FAIMS-MS2 analysis with stepped-HCD fragmentation. Platelets from 3 different patients were each stimulated by thrombin treatment to release their granule contents. These secreted proteins were crosslinked with an optimised DSSO concentration (0.1 mM) and after trypsin digestion were fractionated by offline high pH reversed-phase chromatography to increase crosslinked peptide coverage. MaxQuant was used for crosslinked peptide identification as it showed significant sensitivity improvements compared to XlinkX. Across the 3 patients 1,844 crosslinked peptides were identified from 143 different secreted platelet proteins. For analysis of platelet proteins without confirmed tertiary structures, we generated our own structural predictions with Colabfold (Alphafold2) incorporating knowledge of each protein’s multimerization state. The distance constraints from our crosslinking data were then mapped onto these predicted structures. In general, the crosslinking distance constraints confirmed the predicted structures, which opens the door for these structures to be used for further functional interrogation of these proteins. Regarding protein-protein interactions derived from the interprotein crosslinks we identified, many previously known positive controls were observed such as fibrinogen alpha-beta-gamma trimers. However, many new interactions were also observed and will be the subject of subsequent confirmatory experiments by IP-MS and other methods. One of the unexpected findings was excellent crosslinking data for HDL particles, which are known to associate with platelets. We observed many crosslinks between APOA1 and APOA2, which allowed us for the first time to propose a structure of this key protein complex. The comprehensive human platelet crosslinking dataset provided here will allow identification of novel regulatory mechanisms for drug targeting to address platelet dysfunction and thrombosis.