Platelet secretion and hemostasis require syntaxin-binding protein STXBP5
Shaojing Ye, … , Yoshimi Takai, Sidney W. Whiteheart
Shaojing Ye, … , Yoshimi Takai, Sidney W. Whiteheart
Published October 1, 2014; First published September 17, 2014
Citation Information: J Clin Invest. 2014;124(10):4517-4528. https://doi.org/10.1172/JCI75572.
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Research Article

Platelet secretion and hemostasis require syntaxin-binding protein STXBP5

Abstract

Genome-wide association studies (GWAS) have linked genes encoding several soluble NSF attachment protein receptor (SNARE) regulators to cardiovascular disease risk factors. Because these regulatory proteins may directly affect platelet secretion, we used SNARE-containing complexes to affinity purify potential regulators from human platelet extracts. Syntaxin-binding protein 5 (STXBP5; also known as tomosyn-1) was identified by mass spectrometry, and its expression in isolated platelets was confirmed by RT-PCR analysis. Coimmunoprecipitation studies showed that STXBP5 interacts with core secretion machinery complexes, such as syntaxin-11/SNAP23 heterodimers, and fractionation studies suggested that STXBP5 also interacts with the platelet cytoskeleton. Platelets from Stxbp5 KO mice had normal expression of other key secretory components; however, stimulation-dependent secretion from each of the 3 granule types was markedly defective. Secretion defects in STXBP5-deficient platelets were confirmed via lumi-aggregometry and FACS analysis for P-selectin and LAMP-1 exposure. Interestingly, STXBP5-deficient platelets had altered granule cargo levels, despite having normal morphology and granule numbers. Consistent with secretion and cargo deficiencies, Stxbp5 KO mice showed dramatic bleeding in the tail transection model and defective hemostasis in the FeCl3-induced carotid injury model. Transplantation experiments indicated that these defects were due to loss of STXBP5 in BM-derived cells. Our data demonstrate that STXBP5 is required for normal arterial hemostasis, due to its contributions to platelet granule cargo packaging and secretion.

Authors

Shaojing Ye, Yunjie Huang, Smita Joshi, Jinchao Zhang, Fanmuyi Yang, Guoying Zhang, Susan S. Smyth, Zhenyu Li, Yoshimi Takai, Sidney W. Whiteheart

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Figure 1

STXBP5 is present in human platelets.

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STXBP5 is present in human platelets. (A) The indicated complexes were p...
(A) The indicated complexes were produced in E. coli using the pRSFDuet plasmid with the S-tag peptide fused to the C terminus of the syntaxin. Complexes were bound to protein S agarose beads. After washing, the complex-bound beads were incubated with solubilized human platelet extracts (Plt. ext.). The beads were washed again, and the proteins were eluted with SDS-PAGE loading buffer. The eluted proteins were separated by SDS-PAGE and stained with Sypro Ruby. The specific interacting proteins (T1–T5) were excised, subjected to trypsin digestion, and analyzed by mass spectrometry. The spectra were searched using MASCOT software (see Supplemental Table 1 for search results). (B) The indicated complexes were produced in E. coli using the pRSFDuet plasmid with the S-tag peptide fused to the C terminus of the syntaxin. Solubilized human platelet extracts were incubated with the different complexes, and the resulting material was recovered on protein S agarose beads. After washing, the proteins were eluted and probed with anti-STXBP5 mAb. (C) Analysis of STXBP5 isoforms by RT-PCR. The human platelet–derived mRNA was converted to cDNA and preamplified by using either random hexamers or oligo d(T)16 primers, followed by regular PCR using isoform-specific primers for s-STXBP5, m-STXBP5, and b-STXBP5 (see Methods). The 1-kb DNA ladder standard is at the far left.