BackgroundPannexin-1 (Panx1) forms an anion-selective channel with a permeability up to ∼1 kDa and represents a non-lytic, non-vesicular ATP release pathway in erythrocytes, leukocytes and neurons. Related connexin gap junction proteins have been reported in platelets; however, the expression and function of the pannexins remain unknown.ObjectiveTo determine the expression and function of pannexins in human plate-lets, using molecular, cellular and functional techniques.MethodsPanx1 expression in human platelets was det-ermined using qPCR and antibody-based techniques. Contributions of Panx1 to agonist-evoked efflux of cytoplasmic calcein, Ca2+ influx, ATP release and aggregation were assessed in washed platelets under conditions where the P2X1 receptor response was preserved (0.32 U mL−1 apyrase). Thrombus formation in whole blood was assessed in vitro using a shear chamber assay. Two structurally unrelated and widely used Panx1 inhibitors, probenecid and carbenoxolone, were used throughout this study, at concentrations that do not affect connexin channels.ResultsPANX1, but not PANX2 or PANX3, mRNA was detected in human platelets. Furthermore, Panx1 protein is glycosylated and present on the plasma membrane of platelets, and displays weak physical association with P2X1 receptors. Panx1 inhibition blocked thrombin-evoked efflux of calcein, and reduced Ca2+ influx, ATP release, platelet aggregation and thrombus formation under arterial shear rates in vitro. The Panx1-dependent contribution was not additive to that of P2X1 receptors.ConclusionsPanx1 is expressed on human platelets and amplifies Ca2+ influx, ATP release and aggregation through the secondary activation of P2X1 receptors. We propose that Panx1 represents a novel target for the management of arterial thrombosis.

Following platelet adhesion and primary activation at sites of vascular injury, secondary platelet activation is induced by soluble platelet agonists, such as ADP, ATP, thrombin and thromboxane. Zinc ions are also released from platelets and damaged cells and have been shown to act as a platelet agonist. However, the mechanism of zinc-induced platelet activation is not well understood. Here we show that exogenous zinc gains access to the platelet cytosol and induces full platelet aggregation that is dependent on platelet protein tyrosine phosphorylation, PKC and integrin αIIbβ3 activity and is mediated by granule release and secondary signalling. ZnSO4 increased the binding affinity of GpVI, but not integrin α2β1. Low concentrations of ZnSO4 potentiated platelet aggregation by collagen-related peptide (CRP-XL), thrombin and adrenaline. Chelation of intracellular zinc reduced platelet aggregation induced by a number of different agonists, inhibited zinc-induced tyrosine phosphorylation and inhibited platelet activation in whole blood under physiologically relevant flow conditions. Our data are consistent with a transmembrane signalling role for zinc in platelet activation during thrombus formation.

Clinical use of tissue plasminogen activator (tPA) in thrombolytic therapy is limited by its short circulation time and hemorrhagic side effects. Inspired by fibrinogen binding to activated platelets, we report a fibrinogen-mimicking, multiarm nanovesicle for thrombus-specific tPA delivery and targeted thrombolysis. This biomimetic system is based on the lipid nanovesicle coated with polyethylene glycol (PEG) terminally conjugated with a cyclic RGD (cRGD) peptide. Our experiments with human blood demonstrated its highly selective binding to activated platelets and efficient tPA release at a thrombus site under both static and physiological flow conditions. Its clot dissolution time in a microfluidic system was comparable to that of free tPA. Furthermore, we report a purpose-built computational model capable of simulating targeted thrombolysis of the tPA-loaded nanovesicle and with a potential in predicting the dynamics of thrombolysis in physiologically realistic scenarios. This combined experimental and computational work presents a promising platform for development of thrombolytic nanomedicines.

Platelets are the primary cellular determinants of haemostasis and pathological thrombus formation leading to myocardial infarction and stroke. Following vascular injury or atherosclerotic plaque rupture, platelets are recruited to sites of damage and undergo activation induced by a variety of soluble and/or insoluble agonists. Platelet activation is a multi-step process culminating in the formation of thrombi, which contribute to the haemostatic process. Zinc (Zn(2+)) is acknowledged as an important signalling molecule in a diverse range of cellular systems, however there is limited understanding of the influence of Zn(2+) on platelet behaviour during thrombus formation. This review evaluates the contributions of exogenous and intracellular Zn(2+) to platelet function and assesses the potential pathophysiological implications of Zn(2+) signalling. We also provide a speculative assessment of the mechanisms by which platelets could respond to changes in extracellular and intracellular Zn(2+) concentration.

In a randomized comparison of nevirapine or abacavir with zidovudine plus lamivudine, routine viral load monitoring was not performed, yet 27% of individuals with viral failure at week 48 experienced resuppression by week 96 without switching. This supports World Health Organization recommendations that suspected viral failure should trigger adherence counseling and repeat measurement before a treatment switch is considered.

Background  Zinc (Zn 2+ ) is an essential trace element that regulates intracellular processes in multiple cell types. While the role of Zn 2+ as a platelet agonist is known, its secondary messenger activity in platelets has not been demonstrated. Objectives  This article determines whether cytosolic Zn 2+ concentrations ([Zn 2+ ] i ) change in platelets in response to agonist stimulation, in a manner consistent with a secondary messenger, and correlates the effects of [Zn 2+ ] i changes on activation markers. Methods  Changes in [Zn 2+ ] i were quantified in Fluozin-3 (Fz-3)-loaded washed, human platelets using fluorometry. Increases in [Zn 2+ ] i were modelled using Zn 2+ -specific chelators and ionophores. The influence of [Zn 2+ ] i on platelet function was assessed using platelet aggregometry, flow cytometry and Western blotting. Results  Increases of intra-platelet Fluozin-3 (Fz-3) fluorescence occurred in response to stimulation by cross-linked collagen-related peptide (CRP-XL) or U46619, consistent with a rise of [Zn 2+ ] i . Fluoresence increases were blocked by Zn 2+ chelators and modulators of the platelet redox state, and were distinct from agonist-evoked [Ca 2+ ] i signals. Stimulation of platelets with the Zn 2+ ionophores clioquinol (Cq) or pyrithione (Py) caused sustained increases of [Zn 2+ ] i , resulting in myosin light chain phosphorylation, and cytoskeletal re-arrangements which were sensitive to cytochalasin-D treatment. Cq stimulation resulted in integrin α IIb β 3 activation and release of dense, but not α, granules. Furthermore, Zn 2+ -ionophores induced externalization of phosphatidylserine. Conclusion  These data suggest that agonist-evoked fluctuations in intra-platelet Zn 2+ couple to functional responses, in a manner that is consistent with a role as a secondary messenger. Increased intra-platelet Zn 2+ regulates signalling processes, including shape change, α IIb β 3 up-regulation and dense granule release, in a redox-sensitive manner.

Zidovudine/lamivudine/tenofovir has good virological efficacy in advanced HIV disease. In this population, who were infected with HIV-1 subtypes A, C or D, M184V with or without NAMs was the most common route to resistance, whereas K65R was identified less often.

Summary.  Plasmin, the direct fibrinolytic enzyme, was compared with tissue plasminogen activator (t‐PA) in an in vitro thrombolysis model. Plasmin has been prepared in a highly pure form from human plasma and has been stabilized against auto‐degradation by low‐pH formulation. This acidified formulation of plasmin has been designed to have a low buffering capacity so that it can be directly infused into clots in a stable and latently active form. This low‐pH formulation has been shown to be equivalent to a neutral‐pH formulation of plasmin in its extent of clot lysis. An in vitro model of catheter‐assisted thrombolysis has been devised in which large (12 × 0.6 cm), retracted clots are treated with an intrathrombus thrombolytic agent via a multi‐sideport catheter. Plasmin dissolves these plasminogen‐deficient clots in a dose‐dependent manner and is clearly superior to t‐PA. In this model system, t‐PA exhibits efficacy only when retracted clots are replenished with plasminogen.