Role and expression of thrombin receptor PAR-1 in muscle cells and neuromuscular junctions during the synapse elimination period in the neonatal rat.

A role for thrombin and its receptor (ThR) during mammalian skeletal muscle cell differentiation and neuromuscular junction (NMJ) formation has been suggested. Previously, we found that the synapse elimination process in the neonatal rat muscle was accelerated by thrombin and blocked by hirudin, its specific inhibitor (Lanuza et al. [2001] J. Neurosci. Res. 63:330-340). To test whether this process resulted from a signal transduction cascade initiated by activation of ThR, in particular PAR-1, we applied to the levator auris longus (LAL) muscle of newborn rats two synthetic peptides (SFLL and FSLL). SFLL is a potent specific agonist for activation of PAR-1, whereas FSLL is an inactive peptide. We have demonstrated that the activation of PAR-1 by SFLL produced acceleration of the presynaptic loss of connections and the postsynaptic maturation of NMJs. Moreover, Western blot analysis showed that PAR-1 was present in the skeletal muscle, and by immunohistochemistry we detected PAR-1 in muscle fibers concentrated in the synaptic area but also in satellite cells. Several lines of evidence suggested that PAR-1 is localized in the postsynaptic membrane: PAR-1 immunofluorescence was concentrated at denervated synaptic sites and was present in the myotube membrane in vitro in the absence of neurons and in dissociated single muscle fibers from which nerve terminals and Schwann cells had been removed. Taken together, these results indicate that thrombin mediates certain stages of activity-dependent synapse elimination in the skeletal muscle and does so through its action on the thrombin receptor PAR-1 localized, at least in part, on the postsynaptic membrane.