Regulation of the incorporation of tissue factor into microparticles by serine phosphorylation of the cytoplasmic domain of tissue factor

Collier, Mary E. W.; Ettelaie, Camille

Abstract

The mechanisms that regulate the incorporation and release of tissue factors (TFs) into cell-derived microparticles are as yet unidentified. In this study, we have explored the regulation of TF release into microparticles by the phosphorylation of serine residues within the cytoplasmic domain of TF. Wild-type and mutant forms of TF, containing alanine and aspartate substitutions at Ser(253) and Ser(258), were overexpressed in coronary artery and dermal microvascular endothelial cells and microparticle release stimulated with PAR2 agonist peptide (PAR2-AP). The release of TF antigen and activity was then monitored. In addition, the phosphorylation state of the two serine residues within the released microparticles and the cells was monitored for 150 min. The release of wild-type TF as procoagulant microparticles peaked at 90 min and declined thereafter in both cell types. The TF within these microparticles was phosphorylated at Ser(253) but not at Ser(258). Aspartate substitution of Ser(253) resulted in rapid release of TF antigen but not activity, whereas TF release was reduced and delayed by alanine substitution of Ser(253) or aspartate substitution of Ser(258). Alanine substitution of Ser(258) prolonged the release of TF following PAR2-AP activation. The release of TF was concurrent with phosphorylation of Ser(253) and was followed by dephosphorylation at 120 min and phosphorylation of Ser(258). We propose a sequential mechanism in which the phosphorylation of Ser(253) through PAR2 activation results in the incorporation of TF into microparticles, simultaneously inducing Ser(258) phosphorylation. Phosphorylation of Ser(258) in turn promotes the dephosphorylation of Ser(253) and suppresses the release of TF.

Citation

Collier, M. E. W., & Ettelaie, C. (2011). Regulation of the incorporation of tissue factor into microparticles by serine phosphorylation of the cytoplasmic domain of tissue factor. Journal of Biological Chemistry, 286(14), 11977-11984. https://doi.org/10.1074/jbc.m110.195214

• Journal Article Type: Article
• Acceptance Date: Feb 8, 2011
• Online Publication Date: Feb 10, 2011
• Publication Date: Apr 8, 2011
• Deposit Date: Nov 13, 2014
• Publicly Available Date: Nov 13, 2014
• Journal: Journal of biological chemistry
• Print ISSN: 0021-9258
• Electronic ISSN: 1083-351X
• Publisher: American Society for Biochemistry and Molecular Biology
• Peer Reviewed: Peer Reviewed
• Volume: 286
• Issue: 14
• Pages: 11977-11984
• DOI: https://doi.org/10.1074/jbc.m110.195214
• Keywords: Coagulation factors; Endothelium; Phospholipid vesicle; Protein phosphorylation; Signal transduction; Endothelial cells; Microparticles; PAR2; Serine phosphorylation; Tissue factor
• Public URL: https://hull-repository.worktribe.com/output/462683
• Publisher URL: http://www.jbc.org/content/286/14/11977
• Additional Information: Copy of article first published in Journal of biological chemistry, 2011, v.286, issue 14