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Lazarus A.H.,Canadian Blood Services | Lazarus A.H.,Li Ka Shing Knowledge Institute | Lazarus A.H.,Toronto Platelet Immunobiology Group | Lazarus A.H.,University of Toronto | And 5 more authors.
Seminars in Hematology | Year: 2013

Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by both accelerated clearance of autoantibody-sensitized platelets and suboptimal platelet production. A number of studies have provided evidence of disturbed innate and adaptive immune responses in patients with ITP. This brief review will highlight some of the more recent work in this field and highlight other findings that provide a potential link between ITP, systemic lupus erythematosus (SLE), and autoimmune hemolytic anemia (AHA). © 2013 Elsevier Inc.

McKenzie C.G.J.,Toronto Platelet Immunobiology Group | McKenzie C.G.J.,St Michaels Hospital | Kim M.,Toronto Platelet Immunobiology Group | Kim M.,St Michaels Hospital | And 7 more authors.
Blood | Year: 2014

Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-related mortality and can occur with any type of transfusion. TRALI is thought to be primarily mediated by donor antibodies activating recipient neutrophils resulting in pulmonary endothelial damage. Nonetheless, details regarding the interactions between donor antibodies and recipient factors are unknown. A murine antibody-mediated TRALI model was used to elucidate the roles of the F(ab″)2 and Fc regions of a TRALI-inducing immunoglobulin G anti-major histocompatibility complex (MHC) class I antibody (34.1.2s). Compared with intact antibody, F(ab″)2 fragments significantly increased serum levels of the neutrophil chemo attractant macrophage inflammatory protein 2 (MIP-2); however, pulmonary neutrophil levels were only moderately increased, and no pulmonary edema or mortality occurred. Fc fragments did not modulate any of these parameters. TRALI induction by intact antibody was completely abrogated by in vivo peripheral blood monocyte depletion by gadolinium chloride (GdCl3) or chemokine blockade with a MIP-2 receptor antagonist but was restored upon repletion with purified monocytes. The results suggest a two-step process for antibody-mediated TRALI induction: the first step involves antibody binding its cognate antigen on blood monocytes, which generates MIP-2 chemokine production that is correlated with pulmonary neutrophil recruitment; the second step occurs when antibody-coated monocytes increase Fc-dependent lung damage. © 2014 by The American Society of Hematology.

Crow A.R.,University of Toronto | Crow A.R.,Toronto Platelet Immunobiology Group | Suppa S.J.,University of Toronto | Suppa S.J.,Toronto Platelet Immunobiology Group | And 6 more authors.
Blood | Year: 2011

To definitively determine whether the neonatal Fc receptor (FcRn) is required for the acute amelioration of immune thrombocytopenia (ITP) by IVIg, we used FcRn-deficient mice in a murine ITP model. Mice injected with antiplatelet antibody in the presence or absence of IVIg displayed no difference in platelet-associated IgG between FcRn deficient versus C57BL/6 mice. FcRn-deficient mice treated with high-dose (2 g/kg) IVIg or a low-dose (2 mg/kg) of an IVIg-mimetic CD44 antibody were, however, protected from thrombocytopenia to an equivalent extent as wild-type mice. To verify and substantiate the results found with FcRn-deficient mice, we used β 2- microglobulin-deficient mice (which do not express functional FcRn) and found that IVIg or CD44 antibody also protected them from thrombocytopenia. These data suggest that for both high-dose IVIg as well as low-dose CD44 antibody treatment in an acute ITP model, FcRn expression is neither necessary nor required. © 2011 by The American Society of Hematology.

Lazarus A.H.,Canadian Blood Services | Lazarus A.H.,Toronto Platelet Immunobiology Group | Lazarus A.H.,University of Toronto
Journal of Clinical Immunology | Year: 2010

Intravenous immunoglobulin (IVIG) is an effective treatment for a variety of autoimmune and inflammatory conditions. The mechanism of action of IVIG remains poorly understood, but a variety of theories have been suggested. Recent studies in murine models have indicated that some of the ameliorative effects of IVIG in autoimmunity can be repeated by the adoptive transfer of leukocytes that have been primed with IVIG. The active cell component within the leukocyte cell population in immune thrombocytopenia (ITP) was determined to be CD11c+ dendritic cells. This review will highlight recent work in murine systems that indicates that the effects of IVIG can be adoptively transferred in some autoimmune diseases and inflammatory states. © Springer Science+Business Media, LLC 2010.

Semple J.W.,Li Ka Shing Knowledge Institute | Semple J.W.,University of Toronto | Semple J.W.,Toronto Platelet Immunobiology Group | Freedman J.,Li Ka Shing Knowledge Institute
Cellular and Molecular Life Sciences | Year: 2010

Although platelets are best known as primary mediators of hemostasis, this function intimately associates them with inflammatory processes, and it has been increasingly recognized that platelets play an active role in both innate and adaptive immunity. For example, platelet adhesive interactions with leukocytes and endothelial cells via P-selectin can lead to several pro-inflammatory events, including leukocyte rolling and activation, production of cytokine cascades, and recruitment of the leukocytes to sites of tissue damage. Superimposed on this, platelets express immunologically-related molecules such as CD40L and Toll-like receptors that have been shown to functionally modulate innate immunity. Furthermore, platelets themselves can interact with microorganisms, and several viruses have been shown to cross-react immunologically with platelet antigens. This review discusses the central role that platelets play in inflammation, linking them with varied pathological conditions, such as atherosclerosis, sepsis, and immune thrombocytopenic purpura, and suggests that platelets also act as primary mediators of our innate defences. © 2009 Birkhäuser Verlag, Basel/Switzerland.

Gyulkhandanyan A.V.,Li Ka Shing Knowledge Institute | Mutlu A.,Li Ka Shing Knowledge Institute | Freedman J.,Li Ka Shing Knowledge Institute | Freedman J.,Toronto Platelet Immunobiology Group | And 5 more authors.
Journal of Thrombosis and Thrombolysis | Year: 2012

Apoptosis, or programmed cell death, is a physiological mechanism that serves for controlled deletion of damaged cells. While long attributed exclusively to nucleated cells, over recent years it has been recognized that apoptosis also occurs in anucleate platelets. We describe here experiences of determining markers of apoptosis in human platelets treated in vitro with proapoptotic chemical and physical stimuli. These include depolarization of mitochondrial inner membrane, cytochrome c release, expression of pro-apoptotic and antiapoptotic proteins of Bcl-2 family, activation of apoptosis executioner caspase-3, exposure of phosphatidylserine, platelet shrinkage, fragmentation to microparticles, blebbing and filopod extrusion on the platelet surface. These assays serve to characterize platelet apoptosis in different cellular compartments (mitochondria, cytosol and plasma membrane) and at the whole-cell level. Methods commonly employed in studies of platelet apoptosis markers include flow cytometry, Western blot analysis and electron microscopy. An integrated methodological approach, based on determination of different platelet apoptosis markers, represents a useful tool for examining platelet apoptosis in various physiological and pathological settings. © Springer Science+Business Media, LLC 2012.

Leytin V.,Li Ka Shing Knowledge Institute | Leytin V.,Toronto Platelet Immunobiology Group | Leytin V.,University of Toronto
Blood Reviews | Year: 2012

For many years, programmed cell death, known as apoptosis, was attributed exclusively to nucleated cells. Currently, however, apoptosis is also well-documented in anucleate platelets. This review describes extrinsic and intrinsic pathways of apoptosis in nucleated cells and in platelets, platelet apoptosis induced by multiple chemical stimuli and shear stresses, markers of platelet apoptosis, mitochodrial control of platelet apoptosis, and apoptosis mediated by platelet surface receptors PAR-1, GPIIbIIIa and GPIbα. In addition, this review presents data on platelet apoptosis provoked by aging of platelets in vitro during platelet storage, platelet apoptosis in pathological settings in humans and animal models, and inhibition of platelet apoptosis by cyclosporin A, intravenous immunoglobulin and GPIIbIIIa antagonist drugs. © 2011 Elsevier Ltd.

Gyulkhandanyan A.V.,Li Ka Shing Knowledge Institute | Mutlu A.,Li Ka Shing Knowledge Institute | Freedman J.,Li Ka Shing Knowledge Institute | Freedman J.,Toronto Platelet Immunobiology Group | And 5 more authors.
British Journal of Haematology | Year: 2013

Anucleate platelets perform two fundamental processes, activation and apoptosis. We elaborated an approach for selective and concurrent stimulation of platelet apoptosis and/or activation, processes important in haemostasis and platelet clearance. Human platelets were treated with BH3 mimetic ABT-737, thrombin, calcium ionophore A23187 and matched diluents. Apoptosis was determined as mitochondrial inner membrane potential (ΔΨm) depolarization and activation as P-selectin exposure. At optimal treatment conditions (90-180 min, 37°C), ABT-737 predominantly induced apoptosis, when 77-81% platelets undergo only ΔΨm depolarization. The ABT-737 impact on ΔΨm depolarization is strongly time- and temperature-dependent, and much higher at 37°C than at room temperature. In contrast, when platelets were treated with thrombin for 15-90 min at either temperature, activation-only was predominantly (79-85%) induced, whereas A23187 triggers both apoptosis and activation (73-81%) when platelets were treated for 15-60 min at 37°C or 15-90 min at room temperature. These data demonstrate that, depending on the triggering stimulus, platelets predominantly undergo ΔΨm depolarization-only, P-selectin exposure-only, or both responses, indicating that platelet apoptosis and activation are different phenomena driven by different mechanisms. The described model provides a basis for studying differential pharmacological manipulation of platelet apoptosis and activation and their role in haemostasis, thrombosis and platelet clearance. © 2013 Blackwell Publishing Ltd.

Mutlu A.,Li Ka Shing Knowledge Institute | Gyulkhandanyan A.V.,Li Ka Shing Knowledge Institute | Freedman J.,Li Ka Shing Knowledge Institute | Freedman J.,Toronto Platelet Immunobiology Group | And 5 more authors.
British Journal of Haematology | Year: 2013

The cell plasma membrane is tightly coupled with the vital processes of apoptosis and activation. In the current study, we investigated exposure of the apoptosis marker phosphatidylserine (PS) and activation marker P-selectin (CD62) on the plasma membrane of anucleate platelets. We found that, depending on triggering stimuli, the plasma membrane of human platelets may exist in four states with predominant exposure of (i) PS but not CD62 (75·9 ± 2·8% of total cells), (ii) CD62 but not PS (86·2 ± 1·3%), (iii) both PS and CD62 (89·6 ± 1·0%) or (iv) neither PS nor CD62 (87·9-97·5%), when platelets were treated at optimal conditions with pro-apoptotic BH3 mimetic ABT-737, thrombin, calcium ionophore A23187 or control diluents, respectively. The dynamics of PS exposure induced by ABT-737 is a slow temperature-dependent process requiring 90 min treatment at 37°C rather than at room temperature for obtaining high levels of PS exposure. In contrast, thrombin-induced CD62 exposure and A23187-induced PS and CD62 exposure showed fast temperature-independent dynamics. This model of selective and concurrent stimulation of PS and/or CD62 transition to the platelet surface provides an experimental horizon for elucidating the roles of plasma membrane markers of platelet apoptosis and activation in platelet clearance. © 2013 John Wiley & Sons Ltd.

Mutlu A.,Li Ka Shing Knowledge Institute | Gyulkhandanyan A.V.,Li Ka Shing Knowledge Institute | Freedman J.,Li Ka Shing Knowledge Institute | Freedman J.,Toronto Platelet Immunobiology Group | And 5 more authors.
British Journal of Haematology | Year: 2012

Platelet apoptosis and activation have been studied in human platelets treated with BH3-only mimetic ABT-737 and calcium ionophore A23187, agents triggering apoptosis through the intrinsic mitochondrial pathway. Platelet apoptosis was determined as activation of crucial apoptosis-associated caspases, initiator caspase-9 of intrinsic apoptosis pathway, executioner caspase-3 and initiator caspase-8 of extrinsic death receptor pathway, and platelet activation was detected by P-selectin (CD62) exposure on the platelet surface. We found that ABT-737 predominantly induced activation of caspases-9, -3 and -8 rather than CD62 exposure, whereas A23187 induces both caspases activation and CD62 exposure. Caspase-8 activation was stimulated independently of the extrinsic apoptosis pathway via mitochondrial membrane permeabilization and depolarization. These data suggest that (i) caspase-8 activation is triggered in ABT-737- and A23187-treated anucleate platelets through the mitochondria-initiated caspase activation cascade bypassing the death receptors, and (ii) ABT-737-treated platelets are a useful experimental tool for discerning the role of platelet apoptosis in platelet function and survival. © 2012 Blackwell Publishing Ltd.

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