2. Academic Validation
  3. Relative distribution and biological characterization of CXCL4L1 isoforms in platelets from healthy donors

Relative distribution and biological characterization of CXCL4L1 isoforms in platelets from healthy donors

  • Biochem Pharmacol. 2017 Dec 1;145:123-131. doi: 10.1016/j.bcp.2017.08.020.
Pieter Ruytinx 1,  Rik Janssens 1,  Nele Berghmans 1,  Mieke Gouwy 1,  Isabelle Ronsse 1,  Sandra Liekens 2,  Paul Proost 1,  Jo Van Damme 1,  Sofie Struyf 3
Affiliations

Affiliations

  • 1 KU Leuven, University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Molecular Immunology, B-3000 Leuven, Belgium.
  • 2 KU Leuven, University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, B-3000 Leuven, Belgium.
  • 3 KU Leuven, University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Molecular Immunology, B-3000 Leuven, Belgium. Electronic address: [email protected].
PMID: 28859966 DOI: 10.1016/j.bcp.2017.08.020
Abstract

CXCL4L1, a platelet-derived ELR-negative CXC chemokine, is a powerful angiostatic and anti-tumoral chemokine. We developed a mass spectrometric assay for the detection of different natural CXCL4L1 isoforms. Using this assay, we identified 4 different CXCL4L1 isoforms in the supernatant of thrombin-stimulated platelets from healthy volunteers: the classical isoform CXCL4L1(1-70), CXCL4L1(-4-70), which probably arises through alternative signal peptide removal and two COOH-terminally truncated isoforms CXCL4L1(1-69) and CXCL4L1(-4-69). CXCL4L1(1-70) was the most abundant isoform, whereas CXCL4L1(-4-70) was detected in 50% of the platelet preparations. Since alterations to the NH2-terminus of chemokines can have severe biological consequences, we investigated the impact of the extension with 4 NH2-terminal Amino acids on the biological activity of CXCL4L1. In vitro, CXCL4L1(-4-70) was as potent as CXCL4L1(1-70) in inhibiting signal transduction and migration of human microvascular endothelial cells towards vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2). In a FITC-conjugated dextran cell permeability assay, both splice variants showed a strong but comparable anti-permeable effect upon VEGF stimulation of the endothelial cell monolayer. In vivo angiogenesis induced by FGF-2 was equally reduced by CXCL4L1(1-70) and CXCL4L1(-4-70). In chemotaxis assays with CXCR3A-transfected cells the CXCL4L1 isoforms both induced migration from 125ng/ml onward. Finally, CXCL4L1(1-70) and CXCL4L1(-4-70) showed the same affinity for heparin. In conclusion, the investigated biological activities of CXCL4L1 are not influenced by the four extra NH2-terminal residues present in the alternatively spliced isoform CXCL4L1(-4-70). Therefore, our results suggest that both isoforms equally interact with the CXCR3A and CXCR3B receptor.

Keywords

Angiogenesis; CXCL4; CXCL4L1; Chemokines; Mass spectrometry; Platelets.

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