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International audience; Fibrinolysis and pericellular proteolysis depend on molecular coassembly of plasminogen and its activator on cell, fibrin, or matrix surfaces. We report here the existence of a fibrinolytic cross-talk mechanism bypassing the requirement for their molecular coassembly on the same surface. First, we demonstrate that, despite impaired binding of Glu-plasminogen to the cell membrane by epsilon-aminocaproic acid (epsilon-ACA) or by a lysine-binding site-specific mAb, plasmin is unexpectedly formed by cell-associated urokinase (uPA). Second, we show that Glu-plasminogen bound to carboxy-terminal lysine residues in platelets, fibrin, or extracellular matrix components (fibronectin, laminin) is transformed into plasmin by uPA expressed on monocytes or endothelial cell-derived microparticles but not by tissue-type plasminogen activator (tPA) expressed on neurons. A 2-fold increase in plasmin formation was observed over activation on the same surface. Altogether, these data indicate that cellular uPA but not tPA expressed by distinct cells is specifically involved in the recognition of conformational changes and activation of Glu-plasminogen bound to other biologic surfaces via a lysine-dependent mechanism. This uPA-driven cross-talk mechanism generates plasmin in situ with a high efficiency, thus highlighting its potential physiologic relevance in fibrinolysis and matrix proteolysis induced by inflammatory cells or cell-derived microparticles.

International audience; Redirecting the splicing machinery through the hybridization of high affinity, RNase H- incompetent oligonucleotide analogs such as phosphoramidate morpholino oligonucleotides (PMO) might lead to important clinical applications. Chemical conjugation of PMO to arginine-rich cell penetrating peptides (CPP) such as (R-Ahx-R)(4) (with Ahx standing for 6-aminohexanoic acid) leads to sequence-specific splicing correction in the absence of endosomolytic agents in cell culture at variance with most conventional CPPs. Importantly, (R-Ahx-R)(4)-PMO conjugates are effective in mouse models of various viral infections and Duchenne muscular dystrophy. Unfortunately, active doses in some applications might be close to cytotoxic ones thus presenting challenge for systemic administration of the conjugates in those clinical settings. Structure-activity relationship studies have thus been undertaken to unravel CPP structural features important for the efficient nuclear delivery of the conjugated PMO and limiting steps in their internalization pathway. Affinity for heparin (taken as a model heparan sulfate), hydrophobicity, cellular uptake, intracellular distribution and splicing correction have been monitored. Spacing between the charges, hydrophobicity of the linker between the Arg-groups and Arg-stereochemistry influence splicing correction efficiency. A significant correlation between splicing correction efficiency, affinity for heparin and ability to destabilize model synthetic vesicles has been observed but no correlation with cellular uptake has been found. Efforts will have to focus on endosomal escape since it appears to remain the limiting factor for the delivery of these splice-redirecting ON analogs.

International audience; Studies have shown that antifibrinolytic (aprotinin, tranexamic acid, epsilon-aminocaproic acid) reduce blood loss in orthopedic surgery. However, most lacked sufficient power to evaluate the efficacy and safety on clinical outcomes. This meta-analysis aims to evaluate whether intravenous antifibrinolytics, when compared with placebo, reduce perioperative allogeneic erythrocyte transfusion requirement in adults undergoing orthopedic surgery and whether it might increase the risk of venous thromboembolism. From MEDLINE, EMBASE, and the Cochrane Controlled Trials Register, the authors identified 43 randomized controlled trials in total hip and knee arthroplasty, spine fusion, musculoskeletal sepsis, or tumor surgery performed to July 2005 (for aprotinin, 23 trials with 1,268 participants; tranexamic acid, 20 with 1,084; epsilon-aminocaproic acid, 4 with 171). Aprotinin and tranexamic acid reduced significantly the proportion of patients requiring allogeneic erythrocyte transfusion according to a transfusion protocol. The odds ratio was 0.43 (95% confidence interval, 0.28-0.64) for aprotinin and 0.17 (0.11-0.24) for tranexamic acid. Results suggest a dose-effect relation with tranexamic acid. Epsilon-aminocaproic acid was not efficacious. Unfortunately, data were too limited for any conclusions regarding safety. Although the results suggest that aprotinin and tranexamic acid significantly reduce allogeneic erythrocyte transfusion, further evaluation of safety is required before recommending the use of antifibrinolytics in orthopedic surgery.