Abstract 976 Annexin A7 is a critical regulator of oxylipin generation and PLCγ2-dependent Ca<SUP>2+</SUP> mobilization during platelet activation

Clin Res Cardiol (2021) DOI DOI https://doi.org/10.1007/s00392-021-01843-w
Annexin A7 is a critical regulator of oxylipin generation and PLCγ2-dependent Ca²⁺ mobilization during platelet activation
M.-C. Manke¹, S. Geue¹, B. Peng², F. Kollotzek¹, P. Münzer¹, B. Walker-Allgaier¹, S. Huber³, D. Rath¹, A. Sickmann², D. Dürschmied⁴, F. Lang⁵, M. Gawaz¹, R. Ahrends⁶, O. Borst¹
¹Innere Medizin III, Kardiologie und Kreislauferkrankungen, Universitätsklinikum Tübingen, Tübingen; ²Leibniz-Institut für Analytische Wissenschaften - ISAS, Dortmund; ³Department of Radiation Oncology, University of Tübingen, Tübingen; ⁴Klinik für Kardiologie und Angiologie I, Universitäts-Herzzentrum Freiburg - Bad Krozingen GmbH, Freiburg im Breisgau; ⁵Department of Physiology, University of Tübingen, Tübingen; ⁶Institute for Lipidomics, University of Vienna, Austria, Wien, AT;
Rationale: Platelet activation after contact to subendothelial collagen leads to acute arterial thrombosis. Annexin A7 (ANXA7) is a phospholipid-binding protein participating in the regulation of intracellular Ca²⁺ and exocytosis. Objective: The present study aimed to determine the role of ANXA7 in platelet Ca²⁺-signaling and lipid metabolism during platelet activation in arterial thrombosis using the ANXA7 inhibitor ABO and gene-targeted mice lacking Anxa7 (Anxa7^-/-). Methods and Results: ANXA7 is strongly expressed in platelets and coronary thrombi of patients with acute myocardial infarction. Functionally, luminescence aggregometry revealed significantly abrogated aggregation and secretion of ABO-treated or Anxa7^-/- platelets as compared to untreated or Anxa7^+/+ platelets respectively following activation with collagen or the GPVI-specific agonist collagen-related peptide (CRP). Further, while thrombus formation on collagen-coated surfaces under high arterial shear rates was significantly diminished in ABO-treated or Anxa7-deficient platelets, and thrombotic vascular occlusion after FeCl₃-induced injury in vivo was blunted, no prolongation of bleeding time was observed in Anxa7^-/- BM-chimeric mice. Fura-2-AM spectrofluorometry unravelled a blunted [Ca²⁺]_i increase in Anxa7^-/- platelets after GPVI stimulation. Due to an abolished PLCg2 phosphorylation, Anxa7^-/-platelets displayed significantly reduced IP₃ production and abrogated intracellular Ca²⁺ mobilization following collagen-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 TxA₂ and 12(S)-HETE. Finally, defective PLCg2 phosphorylation and blunted [Ca²⁺]_i increase in Anxa7^-/- platelets could be rescued by exogenous addition of 12(S)-HETE indicating that ANXA7 is a critical regulator of the platelet 12-lipoxygenase in GPVI-dependent platelet Ca²⁺-signaling during arterial thrombosis. Conclusions: The present study unravels ANXA7 as a regulator of oxylipin metabolism and Ca²⁺-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 anti
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