BIN2 orchestrates platelet calcium signaling in thrombosis and thrombo-inflammation
Julia Volz, … , Markus Sauer, Bernhard Nieswandt
Julia Volz, … , Markus Sauer, Bernhard Nieswandt
Published August 4, 2020
Citation Information: J Clin Invest. 2020;130(11):6064-6079. https://doi.org/10.1172/JCI136457.
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Research Article Cell biology Hematology

BIN2 orchestrates platelet calcium signaling in thrombosis and thrombo-inflammation

Abstract

Store-operated Ca2+ entry (SOCE) is the major route of Ca2+ influx in platelets. The Ca2+ sensor stromal interaction molecule 1 (STIM1) triggers SOCE by forming punctate structures with the Ca2+ channel Orai1 and the inositol trisphosphate receptor (IP3R), thereby linking the endo-/sarcoplasmic reticulum to the plasma membrane. Here, we identified the BAR domain superfamily member bridging integrator 2 (BIN2) as an interaction partner of STIM1 and IP3R in platelets. Deletion of platelet BIN2 (Bin2fl/fl,Pf4-Cre mice) resulted in reduced Ca2+ store release and Ca2+ influx in response to all tested platelet agonists. These defects were a consequence of impaired IP3R function in combination with defective STIM1-mediated SOC channel activation, while Ca2+ store content and agonist-induced IP3 production were unaltered. This severely defective Ca2+ signaling translated into impaired thrombus formation under flow and a protection of Bin2fl/fl,Pf4-Cre mice in models of arterial thrombosis and stroke. Our results establish BIN2 as a central regulator of platelet activation in thrombosis and thrombo-inflammatory disease settings.

Authors

Julia Volz, Charly Kusch, Sarah Beck, Michael Popp, Timo Vögtle, Mara Meub, Inga Scheller, Hannah S. Heil, Julia Preu, Michael K. Schuhmann, Katherina Hemmen, Thomas Premsler, Albert Sickmann, Katrin G. Heinze, David Stegner, Guido Stoll, Attila Braun, Markus Sauer, Bernhard Nieswandt

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Figure 3

Defective IP3R function in Bin2fl/fl,Pf4Cre mice and BIN2 interaction with STIM1 and IP3R.

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Defective IP3R function in Bin2fl/fl,Pf4Cre mice and BIN2 interaction wi...
(A and B) Determination of whole-cell tyrosine phosphorylation pattern in WT and Bin2fl/fl,Pf4Cre platelets upon activation with convulxin (CVX) (A) or rhodocytin (RC) (B). The samples were taken at the indicated time points, lysed, and Western blot analysis was performed with the indicated phospho-specific and pan-antibodies. This blot is representative of 3 independent experiments. (C) Quantification of inositol monophosphate (IP1), a specific metabolite of inositol-1,4,5-trisphosphate (IP3), produced upon activation with the indicated agonist (n = 3). Representative results of 3 independent experiments. (D) Western blot analysis of IP3R expression in WT and Bin2fl/fl,Pf4Cre platelet lysates; GAPDH was used as loading control. (E and F) Ca2+ concentrations in the cytoplasm of WT and Bin2fl/fl,Pf4Cre platelets upon treatment with UV light–inducible IP3 in the (E) absence or (F) presence of extracellular Ca2+ (n = 8). (G) Bin2fl/fl,Pf4Cre platelet lysates were incubated with recombinant BIN2-HIS protein, followed by a purification step with NI-NTA beads. The different fractions were eluted and analyzed by Western blotting using BIN2-, STIM1-, and IP3R-specific antibodies. Representative result of 3 independent experiments. Values are depicted as mean ± SD, and P values were calculated using the Mann-Whitney U test. **P < 0.01. See complete unedited blots in the supplemental material.