Role and Expression of Thrombin Receptor PAR-1 in Muscle Cells and Neuromuscular Junctions During the Synapse Elimination Period in the Neonatal Rat Marı ´a A. Lanuza, 1 * Neus Garcia, 1 Carmen M. Gonza ´lez, 1 Manel M. Santafe ´, 1 Phillip G. Nelson, 2 and Josep Tomas 1 1 Unitat d’Histologia i Neurobiologia (UHN), Facultat de Medicina i Cie `ncies de la Salut, Universitat Rovira i Virgili, Reus (Tarragona), Spain 2 Laboratory of Developmental Neurobiology, NICHD, NIH, Bethesda, Maryland A role for thrombin and its receptor (ThR) during mam- malian skeletal muscle cell differentiation and neuromus- cular junction (NMJ) formation has been suggested. Pre- viously, 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 new- born 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 demon- strated that the activation of PAR-1 by SFLL produced acceleration of the presynaptic loss of connections and the postsynaptic maturation of NMJs. Moreover, West- ern blot analysis showed that PAR-1 was present in the skeletal muscle, and by immunohistochemistry we de- tected PAR-1 in muscle fibers concentrated in the syn- aptic 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 re- moved. 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 local- ized, at least in part, on the postsynaptic membrane. © 2003 Wiley-Liss, Inc. Key words: polyneuronal innervation; PAR-1; thrombin The serine protease thrombin plays a central role in the processes of blood coagulation and thrombosis. Al- though these are its better understood roles, thrombin is a multifunctional protease with diverse cellular effects, in- cluding synaptic and neuromuscular junction (NMJ) de- velopmental plasticity. Thrombin action is mediated through a family of specific cell surface protease-activated receptors (PARs). Three of the four PARs (PAR-1, PAR-3, and PAR-4) are activated by thrombin. PARs are abundantly expressed in the adult rat brain (Suidan et al., 1996; Striggow et al., 2001), and it has been proposed that PAR-1 plays a role in development (Soifer et al., 1994; Niclou et al., 1998) and also is developmentally expressed in rat skeletal muscle (Mbedi et al., 2001). Moreover, thrombin receptor (ThR) mRNA was detected at the maximal level at birth, and then the ThR gene expression was developmentally regulated (Suidan et al., 1996; Kim et al., 1998), although ThR mRNA was detected in the adult skeletal muscle, and the amount increased rapidly (in hours) upon denervation (Kim et al., 1998). Expression of protease nexin-1 (PN-1), the potent naturally occurring serine protease inhibitor, has been detected at the NMJ (Festoff et al., 1991). It is developmentally regulated and remains stably concentrated at the NMJ in the adult (Akaaboune et al., 1998). It was hypothesized that throm- bin modulates synapse elimination during postnatal devel- opment, because PN-1 and the specific thrombin inhibi- tor hirudin reduced the electrical activity-dependent loss of synapses (Liu et al., 1994), and the activation of ThR led to the reduction of synapses in cocultures of spinal ventral horn neurons and myotubes (Jia et al., 1999). Hirudin delayed synapse elimination in vivo (Zoubine et Contract grant sponsor: FISS 2000; Contract grant number: 00/0953; Contract grant sponsor: FISS 2002; Contract grant number: 02/0448. *Correspondence to: M.A. Lanuza, Unitat d’Histologia i Neurobiologia, Facultat de Medicina i Cie `ncies de la Salut, Universitat Rovira i Virgili, Sant Llorenc ¸ 21, 43201 Reus (Tarragona), Spain. E-mail: male@fmcs.urv.es Received 26 August 2002; Revised 16 December 2002; Accepted 17 December 2002 Journal of Neuroscience Research 73:10 –21 (2003) © 2003 Wiley-Liss, Inc.