Analysis of the Healthy Platelet Proteome Identifies a New Form of Domain-Specific O-Fucosylation

• Published in: Molecular & cellular proteomics Vol. 23; no. 2; p. 100717
• Main Authors: Houlahan, Callum B., Kong, Yvonne, Johnston, Bede, Cielesh, Michelle, Chau, The Huong, Fenwick, Jemma, Coleman, Paul R., Hao, Huilin, Haltiwanger, Robert S., Thaysen-Andersen, Morten, Passam, Freda H., Larance, Mark
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-02-2024; American Society for Biochemistry and Molecular Biology
• Subjects: EMI domain; fucose; glycosylation; human; platelet; secretion; secretome; PAR; GRN; PTM; MS/MS; RBC; SDC; FDR; MDK; POFUT1; LFQ; WBC; ESI; human; fucose; EMI; ACD; THBS; EGF; MS; HCD; glycosylation; ACN; PGC; EThcD; NCE; EMI domain; TSR; secretion; LTBP; MMRN; platelet; secretome
• ISSN: 1535-9476; 1535-9484
• DOI: 10.1016/j.mcpro.2024.100717

Platelet activation induces the secretion of proteins that promote platelet aggregation and inflammation. However, detailed analysis of the released platelet proteome is hampered by platelets’ tendency to preactivate during their isolation and a lack of sensitive protocols for low abundance releasate analysis. Here, we detail the most sensitive analysis to date of the platelet releasate proteome with the detection of >1300 proteins. Unbiased scanning for posttranslational modifications within releasate proteins highlighted O-glycosylation as being a major component. For the first time, we detected O-fucosylation on previously uncharacterized sites including multimerin-1 (MMRN1), a major alpha granule protein that supports platelet adhesion to collagen and is a carrier for platelet factor V. The N-terminal elastin microfibril interface (EMI) domain of MMRN1, a key site for protein–protein interaction, was O-fucosylated at a conserved threonine within a new domain context. Our data suggest that either protein O-fucosyltransferase 1, or a novel protein O-fucosyltransferase, may be responsible for this modification. Mutating this O-fucose site on the EMI domain led to a >50% reduction of MMRN1 secretion, supporting a key role of EMI O-fucosylation in MMRN1 secretion. By comparing releasates from resting and thrombin-treated platelets, 202 proteins were found to be significantly released after high-dose thrombin stimulation. Complementary quantification of the platelet lysates identified >3800 proteins, which confirmed the platelet origin of releasate proteins by anticorrelation analysis. Low-dose thrombin treatment yielded a smaller subset of significantly regulated proteins with fewer secretory pathway enzymes. The extensive platelet proteome resource provided here (larancelab.com/platelet-proteome) allows identification of novel regulatory mechanisms for drug targeting to address platelet dysfunction and thrombosis.
[Display omitted]
•Human platelet lysate and releasate proteomes were characterized in detail.•>200 proteins were significantly released after high-dose thrombin stimulation.•O-Glycosylation was detected as a dominant modification of the secreted proteins.•A new form of domain-specific O-fucosylation was detected on the protein MMRN1.
The human platelet lysate and releasate proteome was examined using an ultrasensitive protocol. By comparing releasates from resting and thrombin-treated platelets, 202 proteins were found to be significantly released after high-dose thrombin stimulation. Unbiased scanning for posttranslational modifications within releasate proteins highlighted O-glycosylation as being a major component. Elastin microfibril interface domain-specific O-fucosylation was detected on the protein MMRN1 and was demonstrated to be important for its secretion.