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.
View: Text | PDF
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

×

Figure 2

STXBP5 is associated with platelet SNARE complexes and cytoskeleton.

Options: View larger image (or click on image) Download as PowerPoint
STXBP5 is associated with platelet SNARE complexes and cytoskeleton. (A–...
(A–D) Platelet extracts (Input) from resting (R) or thrombin-stimulated (S) platelets were prepared by solubilization with 1% Triton X-100. After clarification, platelet extracts were incubated with anti-STXBP5 mAb (A) or rabbit polyclonal Ab (B and D), anti-STX11 rabbit polyclonal Ab (C), anti-Munc18b goat polyclonal Ab (D), or IgG control for 3 hours at 4°C. Immune complexes were recovered with protein A and G sepharose. The bound proteins were eluted and separated by SDS-PAGE, followed by IB with the indicated antibodies. (E) Washed platelets (2.5 × 108) were resuspended in HEPES/Tyrode buffer and incubated with (stimulated) or without (resting) 0.1 U/ml thrombin for 5 minutes. After disruption by freeze-thaw, the unbroken cells were removed by centrifugation at 700 g for 5 minutes, and supernatants were subjected to ultracentrifugation (100,000 g for 1 hour at 4°C). The cytosolic fractions (S1) were collected. The remaining pellets were solubilized sequentially with Triton X-100 (S2), then n-octyl-β-d-glucopyranoside (S3). The supernatants and insoluble pellet (P) were analyzed by IB with the indicated antibodies. T, total extract (starting material for the fractionation).