O005 Platelet expression of MIF and its secretion by thrombogenic stimulation

Abstract

Atherosclerosis, a chronic inflammatory disease of the arterial wall, is the main underlying pathology of cardiovascular diseases in the Western world. Chemokines in conjunction with leukocytes recruited into the atherogenic vessel wall are key players in this process. It also turned out that platelets have an important impact on the course of the disease, for example through the secretion of a variety of growth factors and inflammatory chemokines such as platelet factor 4 (PF4/CXCL4), RANTES/CCL5, or SDF-1a/CXCL12 [1]. Despite this knowledge, the precise contributions of the involved chemokines are incompletely understood and it has been assumed that platelets contain additional inflammatory factors. We recently demonstrated that the chemokine-like function chemokine macrophage migration inhibitory factor (MIF) has a key role in atherogenic lesion development. MIF is secreted from atherogenic endothelium and activated macrophages and promotes monocyte and T cell recruitment through non-cognate interaction with CXC chemokine receptors [2]. Here we asked whether platelets express MIF and whether platelet-derived MIF would contribute to atherogenic plaque development. Detection and quantification of postulated MIF stores in platelets was performed by Western Blot analysis and ELISA. The presence of MIF in K652 cells differentiated into megakaryocytes was studied by confocal microscopy. MIF secretion was measured after activation of purified human platelets with different stimuli and MIF content in platelet supernatants was quantified by human MIF ELISA. qPCR was used to determine mRNA contents in the megakaryocytic cell line and in primary human platelets. We were able to demonstrate the presence of appreciable amounts of MIF protein in both human and murine platelets. Furthermore, the K562 megakaryocyte line stained positive for MIF. We quantified the amount of MIF in human platelets to be around 0.3 fg MIF/platelet. Moreover, qPCR demonstrated the presence of MIF mRNA in differentiated K562 cells and, although at a low copy number, even in primary human platelets.To begin to examine whether platelet MIF might contribute to local inflammatory processes such as in the atherogenic vessel, MIF secretion from purified human platelets in response to various stimuli was measured by ELISA. After thrombin stimulation, 70% of the endogenous MIF was secreted compared to unstimulated platelets as early as 4 h after stimulation. Likewise, collagen stimulation led to a significant MIF release (paired Student’s t-test; significance: p < 0.05). In contrast to these well-known thrombogenic stimuli, potential atherogenic stimuli like oxLDL or TNF-a did not lead to measurable MIF levels in the platelet supernatants. This is the first report demonstrating MIF secretion from platelets upon thrombogenic activation, suggesting that platelets could be a previously unrecognized source of MIF in the local atherogenic environment of an inflamed vessel.