EDA-Fibronectin Is Dispensable for An Efficient Bone Marrow Thrombopoiesis, but Affects Platelet Size in Mice
Authors: Malara Alessandro , Gruppi Cristian , Muro Andrés , Marco Luigi De , Balduini Alessandra 
Published: October 2019

Abstract
Abstract 2388

BACKGROUND:
Extracellular matrix proteins in the bone marrow concur to the generation of niche microenvironment that regulates stem cells differentiation into mature megakaryocytes (MK) and platelet release in the bloodstream. Among them, fibronectin (FN) has been involved in the regulation of MK maturation and proplatelet release. FN is a multimeric glycoprotein playing important roles in cell adhesion, migration, growth and differentiation. FN gene generates protein diversity as a consequence of alternative processing of a single transcript at three different sites: the Extra Domain A (EDA), the Extra Domain B and the type III connecting segment (IIICS). The EDA is absent from soluble plasma FN but included in the cellular form of FN deposited as insoluble fibrils within tissues. We have recently demonstrated that human cord blood derived MKs express EDA+ FN, fibronectins are important in mediating MK collagen-dependent behavior and MKs are involved in the process of FN fibrillogenesis within bone marrow environment. Further, circulating platelets contain a small amount of MK-derived EDA fibronectin and EDA+ FN has prothrombotic activity in mice. However, the role of FN and its variants in normal thrombopoiesis is still unraveled.

METHODS:
Mouse strains constitutively including (EDA+/+) or excluding (EDA−/−) the EDA domain in all tissues and plasma were used. Primary mouse megakaryocytes were differentiated in vitro from bone marrow and used for RNA isolation, immunofluorescence, fibrinogen adhesion assay and flow cytometry analysis. Femurs of 8 months old mice were fixed, decalcified and frozen sections of 8 microns were stained in immunoistochemistry.

RESULTS:
Modification of EDA+ FN expression in all mouse strains was demonstrated in murine MKs by RT-PCR and immunofluorescence analysis. The presence or the absence of EDA+ FN in murine MKs did not affect platelet count (WT 1462±294 vs EDA+/+ 1273±131 vs EDA−/− 1365±207 103/ml), while both mutant strains presented a higher Mean Platelet Volume (MPV) (WT 7,7±0,3 vs EDA+/+ 8,1±0,2 vs EDA−/− 8,2±0,3 fL n=12 for wt, n=11 for +/+ and −/−; p<0.01 for +/+ and −/− vs WT, respectively; no differences between +/+ and −/−). The Platelet Distribution Width (PDW) was also different between WT and mutants. Further, preliminary results on MK bone marrow content demonstrated similar results in all genotypes (WT 1,29% vs EDA+/+ 1,33% vs EDA−/− 1,36%). MKs differentiated in vitro, from bone marrow haemopoietic progenitors, showed a comparable ploidy levels and ability to extend proplatelets on fibrinogen (WT 3,72% vs EDA+/+ 4,6% vs EDA−/− 3,12%) in all the different genotypes. Bone marrow histology revealed similar architecture after ematoxylin and eosin stain, similar FN distribution in immunofluorescence and no signs of fibrosis independently of EDA inclusion as demonstrated by Masson's trichrome stain.

DISCUSSION:
Although EDA+ FN expression has been demonstrated in human and murine MKs, mouse strains that can't undergo regulated splicing of the EDA exon showed no signs of altered thrombopoiesis, but differences in peripheral platelet dimension. Therefore EDA+ FN may play a role as a platelet dimension determinant during their release: this is an unknown function that requires further investigations.

Disclosures:
No relevant conflicts of interest to declare.