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Anticoagulant therapy-associated bleeding and pathological thrombosis pose serious risks to hospitalized patients. Both complications could be mitigated by developing new therapeutics that safely neutralize anticoagulant activity and inhibit activators of the intrinsic blood clotting pathway, such as polyphosphate (polyP) and extracellular nucleic acids. The latter strategy could reduce the use of anticoagulants, potentially decreasing bleeding events. However, previously described cationic inhibitors of polyP and extracellular nucleic acids exhibit both nonspecific binding and adverse effects on blood clotting that limit their use. Indeed, the polycation used to counteract heparin-associated bleeding in surgical settings, protamine, exhibits adverse effects. To address these clinical shortcomings, we developed a synthetic polycation, Universal Heparin Reversal Agent (UHRA), which is nontoxic and can neutralize the anticoagulant activity of heparins and the prothrombotic activity of polyP. Sharply contrasting protamine, we show that UHRA does not interact with fibrinogen, affect fibrin polymerization during clot formation, or abrogate plasma clotting. Using scanning electron microscopy, confocal microscopy, and clot lysis assays, we confirm that UHRA does not incorporate into clots, and that clots are stable with normal fibrin morphology. Conversely, protamine binds to the fibrin clot, which could explain how protamine instigates clot lysis and increases bleeding after surgery. Finally, studies in mice reveal that UHRA reverses heparin anticoagulant activity without the lung injury seen with protamine. The data presented here illustrate that UHRA could be safely used as an antidote during adverse therapeutic modulation of hemostasis., (© 2017 by The American Society of Hematology.)

The Nef protein of HIV-1 facilitates viral replication and disease progression in vivo. Nef disturbs the organization of immunological synapses between infected CD4(+) T lymphocytes and antigen-presenting B-lymphocytes to interfere with TCR proximal signaling. Paradoxically, Nef enhances distal TCR signaling in infected CD4(+) T lymphocytes, an effect thought to be involved in its role in AIDS pathogenesis. Using quantitative confocal microscopy and cell fractionation of Nef-expressing cells and HIV-1-infected primary human T lymphocytes, we found that Nef induces intracellular compartmentalization of TCR signaling to adjust TCR responses to antigenic stimulation. Nef reroutes kinase-active pools of the TCR signaling master switch Lck away from the plasma membrane (PM) to the trans-Golgi network (TGN), thereby preventing the recruitment of active Lck to the immunological synapse after TCR engagement and limiting signal initiation at the PM. Instead, Nef triggers Lck-dependent activation of TGN-associated Ras-Erk signaling to promote the production of the T lymphocyte survival factor IL-2 and to enhance virus spread. Overexpression of the Lck PM transporter Unc119 restores Nef-induced subversions of Lck trafficking and TCR signaling. Nef therefore hijacks Lck sorting to selectively activate TGN-associated arms of compartmentalized TCR signaling. By tailoring T-lymphocyte responses to antigenic stimulation, Nef optimizes the environment for HIV-1 replication.