ANXA7 Regulates Platelet Lipid Metabolism and Ca <sup>2+</sup> Release in Arterial Thrombosis
Mailin-Christin Manke, Sascha Geue, Cristina Coman, Bing Peng, Ferdinand Kollotzek, Patrick Münzer, Britta Walker, Stephan M. Huber, Dominik Rath, Albert Sickmann, David Stegner, Daniel Duerschmied, Florian Läng, Bernhard Nieswandt, Meinrad Gawaz, Robert Ahrends, Oliver Borst
Abstract
Rationale: Platelet activation after contact to subendothelial collagen leads to acute arterial thrombosis. ANXA7 (Annexin A7) is a phospholipid-binding protein participating in the regulation of intracellular Ca 2+ and exocytosis. Objective: The present study aimed to determine the role of ANXA7 in platelet Ca 2+ signaling and lipid metabolism during platelet activation in arterial thrombosis using the ANXA7 inhibitor 6-amino-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine (ABO) and gene-targeted mice lacking Anxa7 ( Anxa7 −/− ). Methods and Results: ANXA7 is strongly expressed in platelets. Functionally, luminescence aggregometry revealed significantly abrogated aggregation and secretion of ABO-treated or Anxa7 −/− platelets when compared with untreated or Anxa7 +/+ platelets after activation with collagen or the GPVI (glycoprotein VI)-specific agonist collagen-related peptide. Furthermore, while both thrombus formation on collagen-coated surfaces under high arterial shear rates in ABO-treated or Anxa7 -deficient whole blood, and thrombotic vascular occlusion after FeCl 3 -induced injury in vivo in Anxa7 −/− bone marrow chimeric mice were significantly diminished, no prolongation of bleeding time was observed in ABO-treated or Anxa7 −/− mice. Fura-2-AM spectrofluorimetry unraveled a blunted [Ca 2+ ] i increase in ABO-treated or Anxa7 −/− platelets after GPVI stimulation. Due to an abolished phospholipase Cγ2 phosphorylation, Anxa7 −/− platelets displayed abrogated intracellular Ca 2+ mobilization following collagen-related peptide-dependent platelet activation. Quantitative lipidomics analysis further revealed that ANXA7 critically affects platelet oxylipin metabolism following GPVI-dependent platelet activation. Anxa7 −/− platelets showed a significantly reduced generation of several bioactive metabolites, particularly thromboxane A 2 and 12(S)-hydroxy-eicosatetraenoic acid. Finally, defective phospholipase Cγ2 phosphorylation and blunted [Ca 2+ ] i increase in Anxa7 −/− platelets could be rescued by exogenous addition of 12(S)-hydroxy-eicosatetraenoic acid, indicating that ANXA7 is a critical regulator of the platelet 12-lipoxygenase in GPVI-dependent platelet Ca 2+ signaling during arterial thrombosis. Conclusions: The present study unravels ANXA7 as a regulator of oxylipin metabolism and Ca 2+ -dependent platelet activation downstream of GPVI. ANXA7 plays an important role in platelet signaling during arterial thrombosis and thus may reflect a promising target for novel antiplatelet strategies.