PDK1 Determines Collagen-Dependent Platelet Ca2+ Signaling and Is Critical to Development of Ischemic Stroke In Vivo

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http://atvb.ahajournals.org/content/early/2016/06/23/ATVBAHA.115.307105.abstract

1. Patrick Münzer,
2. Britta Walker-Allgaier,
3. Sascha Geue,
4. Eva Geuss,
5. Gregor Hron,
6. Dominik Rath,
7. Daniela Eißler,
8. Stefan Winter,
9. Elke Schaeffeler,
10. Monika Meinert,
11. Martin Schaller,
12. Andreas Greinacher,
13. Matthias Schwab,
14. Tobias Geisler,
15. Christoph Kleinschnitz,
16. Florian Lang,
17. Meinrad Gawaz,
18. Oliver Borst

+ Author Affiliations

1. From the Department of Cardiology and Cardiovascular Medicine (P.M., B.W.-A., S.G., D.R., D.E., T.G., M.G., O.B.), Department of Physiology (B.W.-A., F.L.), Department of Evolutionary Biology of Invertebrates, Institute for Evolution and Ecology (M.M.), Department of Dermatology (M.S.), and Department of Clinical Pharmacology (M.S.), University of Tübingen, Tübingen, Germany; Department of Neurology, University of Würzburg, Würzburg, Germany (E.G., C.K.); Institute for Immunology and Transfusion Medicine, University of Greifswald, Greifswald, Germany (G.H., A.G.); and Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart and University of Tübingen, Tübingen, Germany (S.W., E.S., M.S.).

1. Correspondence to Oliver Borst, MD, Department of Cardiology and Cardiovascular Medicine, University of Tuebingen, Otfried Mueller-Str.10, 72076 Tuebingen, Germany. E-mail oliver.borst@med.uni-tuebingen.de; or Meinrad Gawaz, MD, Department of Cardiology and Cardiovascular Medicine, University of Tuebingen, Otfried Mueller-Str.10, 72076 Tuebingen, Germany. E-mail meinrad.gawaz@med.uni-tuebingen.de

Abstract

Objective—Activation of platelets by subendothelial collagen results in an increase of cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and is followed by platelet activation and thrombus formation that may lead to vascular occlusion. The present study determined the role of phosphoinositide-dependent protein kinase 1 (PDK1) in collagen-dependent platelet Ca²⁺ signaling and ischemic stroke in vivo.

Approach and Results—Platelet activation with collagen receptor glycoprotein VI agonists collagen-related peptide or convulxin resulted in a significant increase in PDK1 activity independent of second-wave signaling. PDK1 deficiency was associated with reduced platelet phospholipase Cγ2–dependent inositol-1,4,5-trisphosphate production and intracellular [Ca²⁺]_i in response to stimulation with collagen-related peptide or convulxin. The defective increase of [Ca²⁺]_i resulted in a substantial defect in activation-dependent platelet secretion and aggregation on collagen-related peptide stimulation. Furthermore, Rac1 activation and spreading, adhesion to collagen, and thrombus formation under high arterial shear rates were significantly diminished in PDK1-deficient platelets. Mice with PDK1-deficient platelets were protected against arterial thrombotic occlusion after FeCl₃-induced mesenteric arterioles injury and ischemic stroke in vivo. These mice had significantly reduced brain infarct volumes, with a significantly increased survival of 7 days after transient middle cerebral artery occlusion without increase of intracerebral hemorrhage. Tail bleeding time was prolonged in pdk1^−/− mice, reflecting an important role of PDK1 in primary hemostasis.

Conclusions—PDK1 is required for Ca²⁺-dependent platelet activation on stimulation of collagen receptor glycoprotein VI, arterial thrombotic occlusion, and ischemic stroke in vivo.