PF4 and CXCL4L1, Its Non-Allelic Variant, Follow the Same Alpha Granule Storage Pathway in Megakaryocytic Cells.

Abstract 4020

Poster Board III-956

Secretory platelet proteins such as the chemokine platelet factor 4 (PF4/CXCL4), are stored in platelet alpha granules from which they are released upon platelet activation. Defects in platelet storage of granule proteins lead to the hereditary bleeding disorder, grey platelet syndrome. The mechanisms of granule storage of platelet proteins remain unknown. We have shown that alpha granule storage of PF4 was dependent upon the 10 amino acid-long Leu41-Lys50 β-loop (El Golli et al, J Biol Chem 280:30329-35, 2005). CXCL4L1 (or PF4var) is a non-allelic variant of PF4, with which it shares high homology (67 identical residues over 70 of the mature sequence) including the same Leu41-Lys50 β-loop, and which is also present in human platelet alpha granules. Recently, Lasagni et al (Blood 109:4127-34, 2007), have shown that contrary to PF4 which follows the granule storage pathway, CXCL4L1 followed the constitutive secretory pathway. This was based on transfection studies in the HEK293 cell line, or on primary vascular cells including human microvascular endothelial cells (HMVECs), human coronary smooth muscle cells or T cells. In these conditions CXCL4L1 and PF4 exhibited distinct intracellular localizations as well as differential secretion: while release in the medium was spontaneous for CXCL4L1, it required agonist-mediated cell activation for PF4. The authors hypothesized that the differences in intracellular trafficking and compartmentalization of CXCL4L1 and PF4 may be due to their signal peptides, which exhibit significant sequence divergence (38%) compared to the mature sequences (4%). In contrast, PF4 and CXCL4L1 share 100% homology within the granule targeting Leu41-Lys50 β-loop. To test whether signal peptides or instead, the Leu41-Lys50 β-loop, specified alpha granule targeting in megakaryocytes, we compared PF4 and CXCL4L1 intracellular trafficking in the Dami cell line, a human megakaryocytic cell line (which stores proteins in alpha granules upon differentiation in the presence of the cytokine thrombopoietin and phorbol myristate acetate). PF4 and CXCL4L1, or constructs with swapped signal peptides and/or deleted Leu41-Lys50 β-loops were fused to the fluorescent reporter proteins GFP and mCherry, respectively, and their intracellular trafficking analyzed by laser scanning fluorescence microscopy. When cotransfected in the human megakaryocytic Dami cell line, PF4 and CXCL4L1 colocalized efficiently to the same granules (over 80%). Colocalization with the endogenous alpha granule resident protein von Willebrand factor was also observed. Deletion of the Leu41-Lys50 β-loop prevented alpha granule subcellular localization of both CXCL4L1 and PF4. Importantly, signal peptide swapping between PF4 and CXCL4L1 did not affect their final granule colocalization. Altogether these results suggest 1) that in a megakaryocytic context, PF4 and CXCL4L1 traffick along the same intracellular pathway, including the alpha granule storage pathway in megakaryocytes, 2) that the shared Leu41-Lys50 β-loop is essential to alpha granule storage of either protein, and 3) that the signal peptides of PF4 or CXCL4L1 do not appear to play any role in final alpha granule localization. The differential subcellular localization of PF4 and CXCL4L1 in non megakaryocytic contexts, if confirmed, would suggest that megakaryocytes possess storage mechanisms distinct from other cell types.