Transmembrane signaling through G protein-coupled receptors (GPCRs) controls a diverse array of cellular processes including metabolism, growth, motility, adhesion, neuronal signaling and blood coagulation. The numerous GPCRs and their key roles in both normal physiology and disease have made them the target for more than 50% of all prescribed drugs. GPCR agonists and antagonists act on the extracellular side of the receptors, whereas the intracellular surface has not yet been exploited for development of new therapeutic agents. Here, we demonstrate the utility of novel cell-penetrating peptides, termed 'pepducins', that act as intracellular inhibitors of signal transference from receptors to G proteins. Attachment of a palmitate lipid to peptides based on the third intracellular loop of protease-activated receptor 1 (PAR1) or PAR4 (refs. 3-5) yielded potent inhibitors of thrombin-mediated aggregation of human platelets. Infusion of the anti-PAR4 pepducin into mice extended bleeding time and protected against systemic platelet activation, consistent with the phenotype of PAR4-deficient mice. We show that pepducins might be used to ascertain the physiological roles of GPCRs and rapidly determine the potential therapeutic value of blockade of a particular signaling pathway.

Background-Thrombin is the most potent agonist of platelets and plays a critical role in the development of arterial thrombosis. Human platelets express dual thrombin receptors, protease-activated receptor (PAR) 1 and PAR4; however, there are no therapeutic strategies that effectively target both receptors. Methods and Results-Platelet aggregation studies demonstrated that PAR4 activity is markedly enhanced by thrombin-PAR1 interactions. A combination of bivalirudin (hirulog) plus a novel PAR4 pepducin antagonist, P4pal-i1, effectively inhibited aggregation of human platelets to even high concentrations of thrombin and prevented occlusion of carotid arteries in guinea pigs. Likewise, combined inhibition of PAR1 and PAR4 with small-molecule antagonists and pepducins was effective against carotid artery occlusion. Coimmunoprecipitation and fluorescence resonance energy transfer studies revealed that PAR1 and PAR4 associate as a heterodimeric complex in human platelets and fibroblasts. PAR1-PAR4 cofactoring was shown by acceleration of thrombin cleavage and signaling of PAR4 on coexpression with PAR1. Conclusions-We show that PAR1 and PAR4 form a stable heterodimer that enables thrombin to act as a bivalent functional agonist. These studies suggest that targeting the PAR1-PAR4 complex may present a novel therapeutic opportunity to prevent arterial thrombosis.

Objectives: The objective of this case series is to review our experience with spontaneous pneumomediastinum, review the available literature, and refine the current clinical approach to this uncommon condition.

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Reperfusion after severe crush injury is an infrequent, but life-threatening condition. It is a unique aspect of prehospital medicine that occurs in the presence of emergency responders attempting to extricate and treat patients who have suffered a crushing injury. These events are unlikely to occur in the hospital setting and, as a result, remain poorly studied. Some evidence exists regarding prophylaxis, but the efficacy of these treatments has not been clearly established. The use of commercial tourniquets to delay the onset of reperfusion injury has previously been described in theory. Extensive literature now exists supporting the safety of tourniquet use in limb trauma and this potential life-saving measure requires further study in patients with crush injury. We present a case of prehospital tourniquet application to delay reperfusion injury after crush injury that resulted in a reduction in morbidity and complete limb salvage.