Abstract Number: PB1982
Meeting: ISTH 2020 Congress
Background: Thrombomodulin and the endothelial protein C receptor (EPCR) participate in anticoagulation by binding thrombin and supporting protein C activation. Coagulation control is crucial for the prevention of major ischemic events, but little is known about coagulation protease signaling during angiogenesis following ischemia.
Aims: To study the importance of the endothelial thrombomodulin-EPCR system in ischemia-induced new vessel formation.
Methods: Unilateral hindlimb ischemia was induced in mice with deletions or targeted mutations of thrombomodulin, EPCR or protease activated receptors (PARs), and reperfusion of distal hindlimbs was monitored using Laser Doppler perfusion imaging (LDPI). Neovascularization in gastrocnemius muscles was analyzed using confocal microscopy and flow cytometry. The aortic ring assay was used to study angiogenic sprouting ex vivo. Next Generation Sequencing of primary mouse endothelial cells was performed to determine the molecular mediators involved in proangiogenic EPCR signaling.
Results: Hindlimb reperfusion and angiogenesis were significantly increased in hypercoagulable mice with a thrombomodulin point mutation on a mixed genetic background associated with elevated EPCR expression indicating a role for EPCR in neovascularization. Analysis of mice on a homogenous genetic background showed that deletion of EPCR in Tie2.Cre-positive cells or of the endothelial thrombin receptor PAR1, but not of PAR2 or PAR4, significantly diminished reperfusion following hindlimb ischemia. Revascularization was significantly impaired in mice with a targeted mutation (PAR1 R46Q) preventing PAR1 activation by activated protein C (aPC) and only marginally in those with disruption of the canonical thrombin cleavage site (PAR1 R41Q). Aortic ring assay in combination with TR41 and TR47 peptides confirmed PAR1-dependent angiogenic response upon Arg-46 cleavage by aPC. NGS revealed upregulation of pathways involved in the regulation of eNOS signaling.
Conclusions: In addition to prior studies implicating thrombin signaling as major driver for pathological angiogenesis, our data indicate that neovascularization after peripheral ischemia is controlled by EPCR−aPC−PAR1 signaling in endothelial cells or progenitor populations.
To cite this abstract in AMA style:Bochenek M, Gogiraju R, Orth J, Großmann S, Reyda S, Spronk H, Münzel T, Griffin J, Ruf W, Schäfer K. Endothelial Protein C Receptor Signaling via Protease-activated Receptor 1 Promotes Neovascularization after Ischemia in Mice [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/endothelial-protein-c-receptor-signaling-via-protease-activated-receptor-1-promotes-neovascularization-after-ischemia-in-mice/. Accessed March 4, 2024.
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