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Abstract
Dextran sulfate (DxS; Mr 500 kD) induces fibrinogen receptor (αIIbβ3) activation via CLEC-2/Syk signaling and via a Syk-independent SFK/PI3K/Akt-dependent tyrosine kinase pathway in human and murine platelets. The platelet surface receptor, responsible for the DxS-induced Syk-independent Akt-activation, has hitherto not been identified. We found that DxS elicited a concentration-dependent aggregation of human platelets resulting from direct PEAR1 activation by DxS. Blocking the PEAR1 receptor, in combination with a selective Syk-inhibitor, completely abrogated the DxS-driven platelet aggregation. The DxS-induced Syk-phosphorylation was not affected in Pear1−/− platelets, but Akt-phosphorylation was largely abolished. As a result, the aggregation of Pear1−/− platelets was reduced and reversible, i.e. aggregates were less stable compared to wild-type platelet aggregates. Moreover, DxS-induced Pear1−/− platelet aggregation was fully abrogated by Syk inhibition, indicating that the remaining platelet aggregation of Pear1−/− platelets was Syk dependent. Hence, the Pear1/c-Src/PI3K/Akt- and CLEC-2/Syk-signaling pathways are independently and additively activated during platelet aggregation by DxS. Conclusion. The DxS-induced aggregation of human and murine platelets is the result of activation of PI3K/Akt through direct PEAR1 phosphorylation and parallel Syk-signaling through CLEC-2.
Acknowledgments
The authors thank Dr G. Wright (Cell Surface Signalling Laboratory, Wellcome Trust Sanger Institute, Cambridge, UK) for the design of the sFcεR1α, s5FcεR1α, and s5PEAR1 recombinant protein and appreciate the skillful assistance of Marleen Lox. The Pear1−/− mouse was available through courtesy of the International Knockout Mouse (KOMP)—Consortium; Sanger Institute, Cambridge.
Funding
This work was supported by the “Fonds voor Wetenschappelijk Onderzoek (FWO) Vlaanderen” (CVDB: grant 11F2512N; BI: post-doctoral fellowship 12M2715N; PV: grant 1801414N—senior clinical investigatorship, grant G0A6514N). The Center for Molecular and Vascular Biology is supported by the “Programmafinanciering KU Leuven (PF/10/014)” and by the “Geconcerteerde Onderzoeksacties” (GOA 2009/13) from the University of Leuven. This research was supported by the Bayer Chair in Cardiovascular Medicine, an unrestricted grant provided to PV.
Ethical committee
Human samples were handled according to the Helsinki Declaration. Formal permission was given by the Ethics Committee of the Leuven University Hospitals to use blood from healthy individuals. All animal experimental procedures were approved by the local Ethics Committee of the KU Leuven.
Supplemental material
Supplemental data for this article can be accessed on the publishers’ website.
Declaration of interest
None declared.