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

Abstract

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.

Keywords: EMI domain; fucose; glycosylation; human; platelet; secretion; secretome.

Graphical abstract

Fig. 1

Proteomic analysis of platelet lysates and releasates from healthy donors.A, workflow for platelet isolation and stimulation with thrombin. B, Coomassie stained SDS-PAGE gel to assess for plasma contamination in platelet lysates and releasates. Arrow indicates albumin. C, histograms of platelet activation using PAC-1 intensity (x-axis). Resting platelets are shown in gray, platelets stimulated with either low-dose (0.025 U/ml) or high-dose (0.2 U/ml) thrombin shown in red. D, workflow for lysate/releasate proteomic analysis. E, heat map showing platelet proteins that were significantly increased in the releasate by high-dose thrombin stimulation or not significantly regulated, n = 5. F, boxplots of proteins known to be secreted by platelets after thrombin activation. Each line represents a single donor. The y-axis shows the label-free quantitation (LFQ) intensity for each protein.

Fig. 2

Unbiased detection of protein modifications including O-glycosylation of platelet releasates. Histograms of the open-search analysis of high-dose thrombin stimulated platelet releasates showing the number of peptide spectral matches (PSMs) (y-axis) across all platelet samples identified as having a range of mass adducts (x-axis, each bar represents a 1 Th wide mass adduct bin). PSMs with a Byonic log probability (Log Prob) score >8 and had modifications to S,T,Y,K,R,D,E,N,Q,P,M,W were plotted. Modifications were plotted separately for common low-mass modifications (A) and higher mass modifications (B). Delta masses corresponding to O-glycan masses are indicated by the glycan symbols. C, quantitative O-glycomics analysis of O-glycans detached by β-elimination from platelet releasate proteins as described in Supplementary methods (n = 5). Bond linkage types are indicated in the legend. The identified O-glycans are depicted as relative abundances out of all observed O-glycans (100%). D, boxplots showing quantitative analysis of thrombospondin-1 O-glycosylation at identified sites, (n = 5). W-Man is C-mannose modified tryptophan on the same peptide as the indicated Ser/Thr. E, boxplots showing quantitative analysis of latent-transforming growth factor beta-binding protein 1 O-glycosylation at identified sites, (n = 5). HydroxyN is hydroxylated asparagine on the same peptide as the indicated Ser/Thr. F, boxplots showing quantitative analysis of coagulation factor V O-glycosylation at identified sites, (n = 5).

Fig. 3

Functional grouping and response of platelet proteins significantly increased in the releasate after high-dose thrombin stimulation. Each protein is represented by a circle annotated with the gene name, and the circle color represents the log₂ fold change (thrombin stimulated/resting) in the releasate (i.e., the degree of change). The circle size represents the log₁₀ iBAQ abundance of each protein in the high-dose thrombin stimulated releasate (i.e., the proportion of the protein relative to the total proteins). Stars indicate releasate proteins that were identified to be O-glycosylated.

Fig. 4

Comparison of platelet lysate and releasate proteomes for confident detection of released proteins from thrombin-activated platelets. Scatterplot of proteins significantly regulated in the high-dose (A) and low-dose (B) thrombin-stimulated releasate. Significantly regulated proteins are shown in orange, nonsignificant proteins are shown in blue. The log₂ fold change (thrombin stimulated/resting) for both the each corresponding releasate (x-axis) and the corresponding lysate (y-axis) was used for plotting. C, Venn diagrams indicating the overlap between significantly increased releasate proteins in either the low-dose or high-dose thrombin groups, top. Analysis of the proportion of significantly increased releasate proteins that are annotated by UniProt as secreted, Golgi-associated, lysosome-associated, or other. D, boxplots of proteins significantly regulated in platelet releasates-only (AGA) or lysates-only (CFB) after thrombin activation. Each line represents a single donor. The y-axis shows the label-free quantitation (LFQ) intensity for each protein.

Fig. 5

Identification of a novel O-fucosylation site on platelet multimerin 1.A, electron transfer higher collision energy dissociation (EThcD) mass spectrum of the novel O-fucosylation site at T216 in MMRN1. The intact precursor ions and associated neutral losses are in green, the z and c fragment ion series are shown in blue and red, respectively. B, cartoon of the trimeric MMRN1 structure with key domains shown. Inset, protein sequence alignment of the MMRN1 EMI domain across diverse species. C, structural prediction of the EMI domain from RoseTTafold with cysteine residues shown as ball and stick and the fucosylated threonine residue highlighted inside a pink circle. Cysteine residues are highlighted by showing the side-chain atoms. D, protein sequence alignment of human EMI domains and EGF-like domains that are known to be O-fucosylated by POFUT1. The EMI site is highlighted with a red triangle, indicating the site of O-fucosylation. The EGF-like site is highlighted with the black arrow, indicating the modified residue in EGF-like domains. EGF, epidermal growth factor; EMI, elastin microfibril interface; POFUT1, protein O-fucosyltransferase 1.

Fig. 6

Loss of POFUT1 affects multimerin-1 secretion.A, boxplots of POFUT1 and the negative control RPLP0 (an abundant ribosomal protein) in platelet lysates before and after thrombin activation. Each line represents a single donor. The y-axis shows the label-free quantitation (LFQ) intensity for each protein. B, protein sequence alignment of EMI domains across a wide range of human proteins. The EMI site is highlighted with a red triangle, indicating the site of O-fucosylation. C, top, HEK293T cells were transfected with plasmids encoding MMRN1 WT-Myc, MMRN1 T216A-Myc, MMRN1 T1055A-Myc, or empty vector (EV) and IgG (secretion control). Cells were cultured for 2 days. Cultured medium was collected and analyzed by Western blot probed with anti-Myc and anti-human IgG antibodies. Bottom, the bar graph shows quantified band intensity normalized with IgG bands from three independent transfection experiments (n = 3), plotted as mean ± SD. D, top, HEK-293T cells either WT, POFUT1 KO, or POFUT2 KO were transfected with plasmids encoding MMRN1-Myc, NOTCH1 EGF1-18-Myc, or AdamTS9 TSR2-8-Myc and IgG (secretion control). After 2 days, cultured medium was collected and analyzed by Western blot probed with anti-Myc and anti-human IgG antibodies. Bottom, the bar graph shows quantified band intensity normalized with IgG bands from three independent transfection experiments (n = 3), plotted as mean ± SD. EGF, epidermal growth factor; EMI, elastin microfibril interface; POFUT1, protein O-fucosyltransferase 1; TSR, thrombospondin type 1 repeats.