Platelet secretion and hemostasis require syntaxin-binding protein STXBP5
Shaojing Ye, … , Yoshimi Takai, Sidney W. Whiteheart
Shaojing Ye, … , Yoshimi Takai, Sidney W. Whiteheart
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 7

STXBP5-deficient mouse platelets have a complex granule cargo defect.

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STXBP5-deficient mouse platelets have a complex granule cargo defect. (A...
(A and B) Platelet extracts (5 × 107 platelets/lane) were prepared from 3 individual Stxbp5 KO or WT mice. The indicated proteins were analyzed by IB (A) and quantified with Image Lab 4.0.1 or ImageQuant TL. After normalization using RabGDI as loading control, the Stxbp5 KO/WT ratio was calculated (B). (C) Washed platelets (2 × 106) from 3 individual Stxbp5 KO or WT mice were incubated with FITC-conjugated anti-GP1bβ, anti-αIIbβ3, anti-GPVI, or anti–PECAM-1 for 15 minutes at room temperature. Geometric MFI (mean ± SEM) was determined using flow cytometry. (D) Antibody array analysis of platelet extracts. Extracts were prepared from WT or Stxbp5 KO mice and probed using duplicate Proteome Profiler Mouse Angiogenesis Antibody Arrays (see Methods). The fluorescence intensity from 2 experiments was used to calculate a Stxbp5 KO/WT ratio for each protein detected on the array. The average of the 2 calculated ratios is plotted. Note that the y axis is a log scale. SDF-1, PF4, and angiopoietin 1 were excluded from analysis due to saturation of their respective spots on the array membranes.