Thrombin, an important serine protease, not only plays a pivotal role in platelet aggregation and coagulation, but also through activation of its receptor, seven transmembrane, G-protein-coupled receptor PAR-1, elicits numerous cellular responses in platelets and endothelial cells such as induction of adhesion molecules, production of chemokines, activation of matrix metalloproteinase (MMP)-2, cytoskeletal reorganization and migration. Thrombin is also one of the inflammatory molecules elevated during G-CSF mobilization of hematopoietic stem/progenitor cells (HSPC) and their collection by leukapheresis. We recently reported that components of leukapheresis products including thrombin enhance in vitro chemotaxis of CD34+ cells towards an SDF-1 gradient and in vivo homing to bone marrow (BM) niches in a murine model (
). In this study we investigated whether thrombin enhances the homing-related responses of human HSPC (CD34+ cells) through MMPs, especially membrane-type (MT)1-MMP which is known to be localized on the leading edge of migrating cells and both activates latent proMMPs (MMP-2, -9) and itself has strong pericellular proteolytic activity. We found that stimulation of CD34+ cells with thrombin upregulates mRNA for MT1-MMP and MMP-9 as well as MT1-MMP protein expression (Western blot, flow cytometry) and proMMP-2 and proMMP-9 secretion (zymography). Thrombin was also found to (i) prime trans-Matrigel chemoinvasion of CD34+ cells towards a low SDF-1 gradient (20 ng/mL), which was inhibited by epigallocatechin-3-gallate, a potent inhibitor of MT1-MMP, and (ii) activate MMP-2 in of co-cultures of CD34+ cells with stromal cells (BM fibroblasts and HUVEC) which secrete proMMP-2. We also found that SDF-1 upregulates mRNA and protein expression of MT1-MMP. Moreover, using confocal microscopy we demonstrate for the first time that in CD34+ cells, PAR-1, like CXCR4, is localized in the GM1 fraction of lipid rafts and stimulation of these cells with thrombin as well as SDF-1 increases incorporation of MT1-MMP into membrane lipid rafts. Furthermore, disruption of lipid raft formation by the cholesterol-depleting agent methyl-b-cyclodextrin inhibits MT1-MMP incorporation into membrane lipid rafts and also trans-Matrigel chemoinvasion of CD34+ cells towards SDF-1. Thus we conclude that thrombin, through PAR-1 signalling and the SDF-1-CXCR4 axis, upregulates the incorporation of MT1-MMP into membrane lipid rafts and the interaction of these axes enhances the homing-related responses of HSPC towards SDF-1.
Disclosure: No relevant conflicts of interest to declare.
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