Platelet-Rich Plasma (PRP) is enriched in molecular messengers with restorative effects on altered tissue environments. Upon activation, platelets release a plethora of growth factors and cytokines, either in free form or encapsulated in exosomes, which have been proven to promote tissue repair and regeneration. Translational research on the potential of exosomes as a safe nanosystem for therapeutic cargo delivery requires standardizing exosome isolation methods along with their molecular and morphological characterization. With this aim, we isolated and characterized the exosomes released by human PRP platelets. Western blot analysis revealed that CaCl₂-activated platelets (PLT-Exos-Ca²⁺) released more exosomes than non-activated ones (PLT-Exos). Moreover, PLT-Exos-Ca²⁺ exhibited a molecular signature that meets the most up-to-date biochemical criteria for platelet-derived exosomes and possessed morphological features typical of exosomes as assessed by transmission electron microscopy. Array analysis of 105 analytes including growth factors and cytokines showed that PLT-Exos-Ca²⁺ exhibited lower levels of most analytes compared to PLT-Exos, but relatively higher levels of those consistently validated as components of the protein cargo of platelet exosomes. In summary, the present study provides new insights into the molecular composition of human platelet-derived exosomes and validates a method for isolating highly pure platelet exosomes as a basis for future preclinical studies in regenerative medicine and drug delivery.

Emerging evidence implicates serotonergic descending facilitatory pathways from the brainstem to the spinal cord in the maintenance of pathologic pain. Upregulation of the serotonin receptor 2A (5-HT_2AR) in dorsal horn neurons promotes spinal hyperexcitation and impairs spinal μ-opioid mechanisms during neuropathic pain. We investigated the involvement of spinal glutamate receptors, including metabotropic receptors (mGluRs) and NMDA, in 5-HT_2AR-induced hyperexcitability after spinal nerve ligation (SNL) in rat. High-affinity 5-HT_2AR agonist (4-bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide (TCB-2) enhanced C-fiber-evoked dorsal horn potentials after SNL, which was prevented by mGluR1 antagonist AIDA [(RS)-1-aminoindan-1,5-dicarboxylic acid] but not by group II mGluR antagonist LY 341495 [(2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl)propanoic acid] or NMDA antagonistd-AP5 [d-(−)-2-amino-5-phosphonopentanoic acid]. 5-HT_2AR and mGluR1 were found to be coexpressed in postsynaptic densities in dorsal horn neurons. In the absence of SNL, pharmacological stimulation of 5-HT_2AR with TCB-2 both induced rapid bilateral upregulation of mGluR1 expression in cytoplasmic and synaptic fractions of spinal cord homogenates, which was attenuated by PKC inhibitor chelerythrine, and enhanced evoked potentials during costimulation of mGluR1 with 3,5-DHPG [(RS)-3,5-dihydroxyphenylglycine]. SNL was followed by bilateral upregulation of mGluR1 in 5-HT_2AR-containing postsynaptic densities. Upregulation of mGluR1 in synaptic compartments was partially prevented by chronic administration of selective 5-HT_2AR antagonist M100907 [(R)-(+)-α-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenyl)ethyl]-4-pipidinemethanol], confirming 5-HT_2AR-mediated control of mGluR1 upregulation triggered by SNL. Changes in thermal and mechanical pain thresholds following SNL were increasingly reversed over the days after injury by chronic 5-HT_2AR blockade. These results emphasize a role for 5-HT_2AR in hyperexcitation and pain after nerve injury and support mGluR1 upregulation as a novel feedforward activation mechanism contributing to 5-HT_2AR-mediated facilitation.

Axon initial segments (AISs) and nodes of Ranvier (NRs) are essential regions for saltatory conduction of the action potential along the axon. These two domains are enriched in similar multimolecular complexes, which include voltage-gated sodium channels (Na_v), NF186 (neurofascin 186), NrCAM (neuron glia-related cell adhesion molecule), and cytoskeleton linkers ankyrin G (AnkG) and βIV-spectrin. Identification of novel members of these complexes is critical to better understand their formation, function, and maintenance. Here we report that IQCJ-SCHIP-1, a recently identified isoform of schwannomin-interacting protein-1 (SCHIP-1), is a novel component of both AISs and NRs in the central and peripheral nervous systems. We show that IQCJ-SCHIP-1 binds calmodulin in the absence of Ca²⁺ and is highly enriched at AISs and NRs. IQCJ-SCHIP-1 accumulation at AISs and NRs is a late event, suggesting that IQCJ-SCHIP-1 is likely to play a role in mature AISs and NRs rather than during their formation. IQCJ-SCHIP-1 was not detected at AISs in the absence of AnkG and interacted in vitro with this protein. IQCJ-SCHIP-1 was also absent from central NRs and AISs of quivering mice, which have a mutation of βIV-spectrin. We suggest that IQCJ-SCHIP-1 might participate, along with AnkG and βIV-spectrin, in the stabilization or function of the multimolecular complexes of AISs and NRs, possibly by participating in Ca²⁺-mediated responses.