Several lines of reports have suggested that mature magakaryocytes (MKs) form long cytoplasmic processes containing platelets (PLT) organelles from which PLT break off due to blood flow pressures in bone marrow (BM). These cytoplasmic processes were termed ‘proplatelet'. MKs differentiated from hematopoietic stem cells by in vitro culture also develop similar processes, referred to as ‘proplatelet-like formation (PPF)'. It has been already reported that fibronectin (FN) and phorbol 12-myristate 13-acetate (PMA) are essential for inducing PPF in MKs using CHRF-288 human megakaryoblastic cell line (Jiang F et al. Blood 99, 2002). FN plays important roles in megakaryocytopoiesis through the FN-receptors. The role of adhesive interactions with FN in BM stroma and FN-receptor beta1-integrins has been reported in proliferation, differentiation and maintenance of megakaryocytic lineage cells. However, the substantial role of these FN-receptors and their functional assignment in PPF are not yet fully understood. We first investigated the effects of beta1-integrins on PPF using CHRF-288 cells, which express alpha4beta1-integrin (VLA-4) and alpha5beta1-integrin (VLA-5) as FN-receptors. When the cells were cultured on FN for 3 days, PMA prompted PPF in a dose-dependent manner. While nearly 15% of the cells displayed PPF with PMA (100 ng/mL), no cells cultured with FN alone or PMA alone exhibited PPF. PPF induced by FN plus PMA combination (FN/PMA) was abrogated by addition of anti-alpha4-integrin monoclonal antibodies (mAb) plus anti-alpha5-integrin mAb combination, but not by the addition of anti-alpha4-integrin mAb alone or anti-alpha5-integrin mAb alone. Thus, the adhesive interaction with FN via VLA-4 and VLA-5 were responsible for PPF. We next investigated the effect of TNIIIA2, which enhances the adhesive interaction between FN and beta1-integrins, in PPF induced by FN/PMA. TNIIIA2 (RSTDLPGLKAATHYTITIRGVC) is a 22-mer peptide derived from the 14th FN type III-like (FNIII) repeat in tenascin (TN)-C molecule which we found recently, and it induces the conformational change necessary for functional activation of beta1-integrins (Fukai F et al. J Biol Chem 282, 2007; J Biol Chem 284, 2009). The PPF induced by FN/PMA was highly accelerated when CHRF-288 cells were enforced adhering to FN by treatment with TNIIIA2 (25 microg/mL). More than 45% of the cells displayed PPF with FN/PMA plus TNIIIA2 combination (FN/PMA/TNIIIA2). Blocking experiments using anti-beta1-integrin mAbs indicated that adhesive interaction with FN via VLA-4 and VLA-5 was also responsible for acceleration of PPF induced by FN/PMA/TNIIIA2. On the other hand, control peptide, TNIIIA2mutant (RSTDLPGLKAATHYTATARGVC) did not accelerate PPF induced by PMA/FN. The calculated yield of the cells with PPF induced by FN/PMA/TNIIIA2 was 2.5-fold more than that induced by FN/PMA. We have previously established ‘a three-phase serum-free culture system' to generate large amount of PLT from human cord blood CD34+ cells (Matsunaga T et al. Stem cells 24, 2006). A study on the effect of TNIIIA2 on our ‘three-phase serum-free culture system' is now underway. Finally, we investigated signal transduction pathways responsible for PPF induced by FN/PMA. While FN/PMA induced activation of extracellular signal-regulated protein kinase 1 (ERK1/2), FN alone or PMA alone did not induce ERK1/2 activation. The results was in accordance with the data previously reported by Jiang et at (Blood 99, 2002). TNIIIA2 strongly enhanced activation of ERK1/2 by FN/PMA. However, c-Jun amino-terminal kinase 1 (JNK1), p38 and phosphoinositide-3 kinase (PI3K)/Akt were not stimulated by FN/PMA even in the presence of TNIIIA2. Thus, enhanced activation of ERK1/2 by FN/PMA/TNIIIA2 was responsible for acceleration of PPF by FN/PMA.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.