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bioRxiv · 10.1101/2022.11.01.514776

Ultra-sensitive platelet proteome maps the O-glycosylation landscape and charts the response to thrombin dosage

Houlahan, C.·Kong, Y.·Johnston, B.·Cielesh, M.·Chau, T. H.·Coleman, P.·Hao, H.·Haltiwanger, R. S.·Thaysen-Andersen, M.·Passam, F. H.·Larance, M.
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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 pre-activate 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 >1,300 proteins. Unbiased scanning for post-translational modifications within releasate proteins highlighted O-glycosylation as being a major component. For the first time, we detected O-fucosylation on previously uncharacterised 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 EMI domain of MMRN1, a key site for protein-protein interaction, was O-fucosylated at a conserved threonine within a new consensus sequence. Our data suggest that Protein O-fucosyltransferase 1 (POFUT1) is responsible for this modification. Secretion of MMRN1 was reduced in cells lacking POFUT1, supporting a key role of O-fucosylation in MMRN1 function. 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 >3,800 proteins, which confirmed the platelet origin of releasate proteins by anti-correlation analysis. Low-dose thrombin treatment yielded a smaller subset of significantly regulated proteins with fewer secretory pathway enzymes. The comprehensive platelet proteome resource provided here (larancelab.com/platelet-proteome) allows identification of novel regulatory mechanisms for drug targeting to address platelet dysfunction and thrombosis. Key PointsO_LIHigh-sensitivity proteome mapping of human platelets identifies O-glycosylation of platelet proteins at key functional sites C_LIO_LIPlatelet O-fucosyltransferase POFUT1 regulates the secretion of adhesive protein multimerin-1 (MMRN1) C_LI

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Houlahan, C., Kong, Y., Johnston, B., Cielesh, M., Chau, T. H., Coleman, P., Hao, H., Haltiwanger, R. S., Thaysen-Andersen, M., Passam, F. H., Larance, M.. 2022-11-02. Ultra-sensitive platelet proteome maps the O-glycosylation landscape and charts the response to thrombin dosage. https://doi.org/10.1101/2022.11.01.514776

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