Platelet Casein Kinase 2α is a pivotal regulator of arterial thrombosis and post-ischemic myocardial remodeling

M. Fischer (Tübingen)¹, M. Sigle (Tübingen)¹, M.-C. Manke (Tübingen)¹, J. Marzi (Reutlingen)², J. P. Schütte (Heidelberg)³, N. Sun (Neuherberg)⁴, F. Kollotzek (Tübingen)⁵, M. Zdanyte (Tübingen)⁵, C. Coman (Wien)⁶, D. Heinzmann (Tübingen)⁵, M. Gawaz (Tübingen)⁵, K. Schenke-Layland (Reutlingen)², A. Walch (Neuherberg)⁴, R. Ahrends (Wien)⁶, P. Münzer (Tübingen)¹, O. Borst (Tübingen)^5
1Universitätsklinikum Tübingen Innere Medizin III, Kardiologie und Angiologie Tübingen, Deutschland; ²NMI Naturwissenschaftliches und Medizinisches Institut Reutlingen, Deutschland; ³Universitätsklinikum Heidelberg Klinik für Innere Med. III, Kardiologie, Angiologie u. Pneumologie Heidelberg, Deutschland; ⁴Helmholtz Zentrum Munich Research Unit Analytical Pathology Neuherberg, Deutschland; ⁵Universitätsklinikum Tübingen Innere Medizin III, Kardiologie und Kreislauferkrankungen Tübingen, Deutschland; ⁶Institut für Analytische Chemie Wien, Österreich

Background: Platelets play a major role in thrombo-occlusive diseases such as myocardial infarction. Platelet activation in thrombus formation and cardiovascular thrombo-inflammation depends on Ca²⁺-mediated signaling. Casein Kinase 2 (CK2) is highly expressed in platelets and consists of two regulatory β-subunits linking two catalytic α subunits. Consequently, the present study aimed to dissect the impact of CK2α on platelet activation during arterial thrombosis and platelet-dependent myocardial remodeling following ischemia and reperfusion.

Results: The evaluation of the CK2α-dependent phosphoproteome in platelets from mice with a MK/platelet-specific deletion of Csnk2α1 (Csnk2α1^Pf4Δ/Pf4Δ) and their wildtype littermates (Csnk2α1^lox/lox) revealed regulated phosphosites and potential substrates, including a diminished phosphorylation of the IP₃ receptor type 1 in Csnk2α1^Pf4Δ/Pf4Δ platelets. This finding was verified using immunoblotting and accompanied by attenuated IP₃-induced as well as GPVI-mediated Ca²⁺ mobilization. Functionally, flow cytometric analysis revealed impaired P-selectin exposure and integrin α_IIbβ₃ activation of Csnk2α1^Pf4Δ/Pf4Δ platelets in response to collagen-related peptide (CRP), which was paralleled by decreased platelet spreading on fibrinogen-coated surfaces. In addition, CRP-dependent platelet aggregation and secretion were abrogated in Csnk2α1-deficient platelets. In line with these results, both in vitro thrombus formation and FeCl₃-induced vascular occlusion were defective in Csnk2α1^Pf4Δ/Pf4Δ mice, whereas primary hemostasis was not affected. To further elucidate the role of platelet CK2α for myocardial thrombo-inflammation following ischemia, we subjected Csnk2α1^Pf4Δ/Pf4Δ mice to transient LAD ligation and consecutive reperfusion. We assessed cardiac function by echocardiography and infarct size by Evans blue/triphenyl tetrazolium chloride staining 7 days post-reperfusion. Since Csnk2α1-deficient mice displayed an improved outcome after myocardial ischemia/reperfusion with a significantly improved LV recovery and significantly less ventricular fibrosis, we aimed to determine how platelet CK2 alpha might affect myocardial remodeling. For that purpose we performed spatial metabolomics and multiplexed immunofluorescence imaging using the MACSima™ platform conforming that platelets locally release chondroitinsulfate (CS) and transforming growth factor β (TGF-β) in a CK2α-dependent manner, a finding that was associated with significantly reduced ventricular fibrosis in the myocardial tissue infiltrated with platelets following LAD ligation and reperfusion. Raman spectromics and echocardiography further revealed less fibrotic scar and improved radial and longitudinal strain in hearts of Csnk2α1^Pf4Δ/Pf4Δ mice 7d after myocardial ischemia and reperfusion.

Conclusions: Our results disclose CK2α as pivotal regulator of arterial thrombosis and thrombo-inflammatory processes following ischemia/reperfusion injury. Platelet CK2α is critically involved in the fibrotic scar formation and LV impairment following myocardial ischemia.  Due to its favorable benefit-risk profile, pharmacological targeting of CK2α provides a valuable strategy for the development of new therapies against cardiovascular thrombo-inflammation. Additional studies have to dissect, whether isoform-specific CK2 inhibitors might be superior to currently available ones.