Exploring a role for Tribbles homologue 3 (TRIB3) in platelet function

Abstract

Maintaining physiological haemostasis in the vasculature demands optimal platelet function. The pseudokinase TRIB3 has previously been implicated in the regulation of platelet production, but no published studies have addressed the role of this protein in platelet function. The work presented here aimed to fill this knowledge gap. We identified five rare non-synonymous variants in TRIB3 (predicting p.V107M, p.S146N, p.R149G, p.R153H and p.R181C amino acid substitutions) following analysis of whole exome sequence from 34 patients with unexplained platelet bleeding disorders, who were recruited to the UK Genotyping and Phenotyping of Platelets study. Bioinformatic analysis predicted all five variants to be deleterious, and structural studies using a 3D model of TRIB3 revealed that the amino acid substitutions affected residues that were likely to be located on the protein surface, and thus expected to affect interactions with other proteins. The mass spectrometric analysis showed that all variants caused a gain and loss of interactions with other proteins, including mitochondrial peptides and proteins that have been implicated in platelet activation. In vitro expression showed that whilst wild-type TRIB3 and all five TRIB3 variants localised to the nucleus, the p.V107M, p.R149G and p.R181C variants showed a diffuse expression pattern in contrast to the punctate expression pattern observed for wild-type TRIB3 and the p.S146N and p.R153H variants. Visualisation of the TRIB3/AKT1 protein complex, using a YFP protein complementation assay, revealed four expression patterns, two of which showed subcellular localisation to the cytoplasm, with the cytoplasmic punctate expression pattern of the TRIB3/AKT1 complex co-localising with mitochondria. The R149G, R153H and R181C variants exerted a gain-of-function effect on the interaction with AKT1, but not AKT2. Quantification of a platelet activation marker CD62p showed a gender-specific effect, affecting activation only in platelets from female Trib3-/- mice, and a similar observation was noted for platelet ATP secretion. In summary, our data provide preliminary evidence for the role of TRIB3 in platelet activation and degranulation, and further studies will be necessary to confirm the involvement of TRIB3 in regulating platelet functions, and to correlate the identified rare TRIB3 variants with the observed bleeding phenotypes.