Pig-to-baboon liver xenoperfusion utilizing GalTKO.hCD46 pigs and glycoprotein Ib blockade.

BACKGROUND: Although transplantation of genetically modified porcine livers into baboons has yielded recipient survival for up to 7 days, survival is limited by profound thrombocytopenia, which becomes manifest almost immediately after revascularization, and by subsequent coagulopathy. Porcine von Willebrand's factor (VWF), a glycoprotein that adheres to activated platelets to initiate thrombus formation, has been shown to constitutively activate human platelets via their glycoprotein Ib (GPIb) receptors. Here, we report our pig-to-primate liver xenoperfusion model and evaluate whether targeting the GPIb-VWF axis prevents platelet sequestration. METHODS: Twelve baboons underwent cross-circulation with the following extracorporeal livers: one allogeneic control with a baboon liver, 4 xenogeneic controls with a GalTKO.hCD46 pig liver, 3 GalTKO.hCD46 pig livers in recipients treated with αGPIb antibody during perfusion, and 4 GalTKO.hCD46 pig livers pre-treated with D-arginine vasopressin (DDAVP) in recipients treated with αGPIb antibody during perfusion. RESULTS: All perfused livers appeared grossly and macroscopically normal and produced bile. Xenograft liver perfusion experiments treated with αGPIb antibody may show less platelet sequestration during the initial 2 h of perfusion. Portal venous resistance remained constant in all perfusion experiments. Platelet activation studies demonstrated platelet activation in all xenoperfusions, but not in the allogeneic perfusion. CONCLUSION: These observations suggest that primate platelet sequestration by porcine liver and the associated thrombocytopenia are multifactorial and perhaps partially mediated by a constitutive interaction between porcine VWF and the primate GPIb receptor. Control of platelet sequestration and consumptive coagulopathy in liver xenotransplantation will likely require a multifaceted approach in our clinically relevant perfusion model.

Blocking VWF platelet binding to treat TTP.

Two articles in this issue of Blood from Feys et al and Callewaert et al, respectively, have employed very similar and elegant strategies in attempts to ameliorate the symptoms of thrombotic thrombocytopenic purpura (TTP).

Laser-induced primary and secondary hemostasis dynamics and mechanisms in relation to selective photothermolysis of port wine stains.

BACKGROUND: Superficial vascular anomalies such as port wine stains are commonly treated by selective photothermolysis (SP). The endovascular laser-tissue interactions underlying SP are governed by a photothermal response (thermocoagulation of blood) and a hemodynamic response (thrombosis). Currently it is not known whether the hemodynamic response encompasses both primary and secondary hemostasis, which platelet receptors are involved, and what the SP-induced thrombosis kinetics are in low-flow venules. OBJECTIVES: To (1) define the role and kinetics of primary and secondary hemostasis in laser-induced thrombus formation and (2) determine which key platelet surface receptors are involved in the hemodynamic response. METHODS: 532-nm laser-irradiated hamster dorsal skin fold venules were studied by intravital fluorescence microscopy following fluorescent labeling of platelets with 5(6)-carboxyfluorescein. Heparin and fluorescently labeled anti-glycoprotein Ib-α (GPIbα) and anti-P-selectin antibodies were administered to investigate the role of coagulation and platelet receptors, respectively. Lesional sizes were quantified by software. RESULTS: Laser irradiation consistently produced sub-occlusive thermal coagula. Thrombosis was triggered in all irradiated venules in a thermal coagulum-independent manner and peaked at 6.25min post-irradiation. Heparin decreased the maximum thrombus size and caused thrombosis to reach a maximum at 1.25min. Immunoblocking of GPIbα abated the extent of thrombosis, whereas immunoblocking of P-selectin had no effect. CONCLUSIONS: The hemodynamic response ensues the photothermal response in a thermal coagulum-independent manner and involves primary and secondary hemostasis. Primary hemostasis is mediated by constitutively expressed GPIbα but not by activation-dependent P-selectin.

Fluorescent labeling of platelets with polyanionic fluorescein derivatives.

OBJECTIVE: To investigate whether polyanionic fluorescein derivatives are capable of labeling resting and activated hamster and human platelets. STUDY DESIGN: 5,6-Carboxyfluorescein and calcein were incubated with resting and convulxin-activated hamster and human platelets in the 0-3.4 microM and 0-2.5 microM final concentration range, respectively, and assayed by flow cytometry and confocal microscopy. RESULTS: Resting and activated platelets of both species could be labeled by both fluorophores and quantified by flow cytometry. The fluorescence emission intensity and the fraction of labeled platelets increased linearly with final fluorophore concentration. Examination of labeled platelets by confocal microscopy revealed that 5,6-carboxyfluorescein and calcein colocalized with the platelet glycocalyx and that the fluorophores were often sequestered by the cells. The latter manifested itself by compartmentalization or relatively homogenous fluorophore distribution in the cytosol. CONCLUSION: Hamster and human resting and activated platelets can be fluorescently labeled by the lipophobicfluorescein derivatives 5,6-carboxyfluorescein and calcein as a result of fluorophore avidity to the platelet glycocalyx and sequestration by the cells. Consequently, platelets could be quantified by flow cytometry and visualized by confocal microscopy.

Human platelets produced in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice upon transplantation of human cord blood CD34(+) cells are functionally active in an ex vivo flow model of thrombosis.

Xenotransplantation systems have been used with increasing success to better understand human hematopoiesis and thrombopoiesis. In this study, we demonstrate that production of human platelets in nonobese diabetic/severe combined immunodeficient mice after transplantation of unexpanded cord-blood CD34(+) cells was detected within 10 days after transplantation, with the number of circulating human platelets peaking at 2 weeks (up to 87 x 10(3)/microL). This rapid human platelet production was followed by a second wave of platelet formation 5 weeks after transplantation, with a population of 5% still detected after 8 weeks, attesting for long-term engraftment. Platelets issued from human hematopoietic stem cell progenitors are functional, as assessed by increased CD62P expression and PAC1 binding in response to collagen-related peptide and thrombin receptor-activating peptide activation and their ability to incorporate into thrombi formed on a collagen-coated surface in an ex vivo flow model of thrombosis. This interaction was abrogated by addition of inhibitory monoclonal antibodies against human glycoprotein Ibalpha (GPIbalpha) and GPIIb/IIIa. Thus, our mouse model with production of human platelets may be further explored to study the function of genetically modified platelets, but also to investigate the effect of stimulators or inhibitors of human thrombopoiesis in vivo.

On the interaction of fluorophore-encapsulating PEGylated lecithin liposomes with hamster and human platelets.

Polyethylene glycol (PEG)-grafted phosphatidylcholine liposomes are used as drug carriers due to their low immunogenicity and prolonged circulation time. The interaction between sterically stabilized lecithin liposomes and platelets has not been investigated before, and deserves to be subjected to scrutiny inasmuch as the uptake of liposomes by platelets could be detrimental for drug delivery and primary hemostasis. Consequently, the interaction between resting and convulxin-activated hamster and human platelets and calcein- or 5,6-carboxyfluorescein-encapsulating PEGylated liposomes composed of distearoyl- and dipalmitoyl phosphatidylcholine and PEG-derivatized distearoyl phosphatidylethanolamine was investigated by flow cytometry, confocal microscopy, and a glass capillary thrombosis model. Fluorescently labeled liposomes of the same composition were subsequently assayed in vivo after 15 and 45 min of systemic circulation. Neither resting nor activated hamster and human platelets interacted with liposomes at 0.70 mM lipid concentration. An absence of any interaction was corroborated in the in vivo experiments. Alternatively, flow cytometry assays evinced that human platelets interact with liposomes at lipid concentrations of >or=1.35 mM. These interactions were more profound for activated platelets than resting platelets. We conclude that the use of PEGylated lecithin liposomes at lipid concentrations of <1.35 mM has no detrimental impact on liposomal drug delivery based on PEGylated lecithin liposomes, but that these drug carriers may be associated with a reduced targeting efficacy or compromised primary hemostatic system when used at concentrations of >or=1.35 mM. In contrast, these drug carriers may become valuable in thrombosis- and drug delivery-related research and applications at concentrations of >or=1.35 mM.

Functional genomics in zebrafish permits rapid characterization of novel platelet membrane proteins.

In this study, we demonstrate the suitability of the vertebrate Danio rerio (zebrafish) for functional screening of novel platelet genes in vivo by reverse genetics. Comparative transcript analysis of platelets and their precursor cell, the megakaryocyte, together with nucleated blood cell elements, endothelial cells, and erythroblasts, identified novel platelet membrane proteins with hitherto unknown roles in thrombus formation. We determined the phenotype induced by antisense morpholino oligonucleotide (MO)-based knockdown of 5 of these genes in a laser-induced arterial thrombosis model. To validate the model, the genes for platelet glycoprotein (GP) IIb and the coagulation protein factor VIII were targeted. MO-injected fish showed normal thrombus initiation but severely impaired thrombus growth, consistent with the mouse knockout phenotypes, and concomitant knockdown of both resulted in spontaneous bleeding. Knockdown of 4 of the 5 novel platelet proteins altered arterial thrombosis, as demonstrated by modified kinetics of thrombus initiation and/or development. We identified a putative role for BAMBI and LRRC32 in promotion and DCBLD2 and ESAM in inhibition of thrombus formation. We conclude that phenotypic analysis of MO-injected zebrafish is a fast and powerful method for initial screening of novel platelet proteins for function in thrombosis.

Antiplatelet drugs.

Platelets play a major role in thromboembolic diseases, and so antiplatelet therapy remains crucial in treatment and prophylaxis. Upon vascular injury, platelets rapidly adhere to the exposed subendothelial matrix, after which they become activated, resulting in the recruitment of additional platelets from the circulation to eventually form a stable arterial platelet plug. Although controlled plug formation is desired for the prevention of excessive blood loss and for promoting wound healing, several pathological conditions may result in the formation of occlusive thrombi leading to severe clinical complications, including myocardial infarction and ischaemic stroke. Many antiplatelet approaches have been investigated, interfering with one or more of the different stages in thrombus formation. This review discusses antiplatelet agents that interfere with the three principal phases in thrombus formation: platelet adhesion, amplification of platelet activation and platelet aggregation. For each stage, novel experimental targets and clinically established antiplatelet strategies will be reviewed. Limitations and possible benefits will be discussed for each target.

Inherited traits affecting platelet function.

Inherited platelet disorders constitute a large group of diseases involving a wide range of genetic defects that can lead to bleeding symptoms of varying severity. They are associated with defects in surface membrane glycoproteins resulting in e.g. Bernard Soulier Syndrome and Glanzmann Thrombasthenia causing defects in platelet adhesion and aggregation, respectively, as well as in receptors for agonists (a.o. P2Y(12), TXA(2)) disrupting platelet signalling. Defects affecting platelet granules can be characterised by abnormalities of alpha-granules as in the Gray platelet syndrome or dense granules as in Hermansky-Pudlak and Chediak-Higashi syndromes, the latter two also altering other cytoplasmic organelles such as melanosomes and therefore not restricted to platelets. Finally, defects in proteins essential to signalling pathways (a.o. in Wiskott-Aldrich syndrome) or in platelet-derived procoagulant activity (Scott and Stormorken syndromes) also impair platelet function. For most of the above disorders mouse knockout models have been generated, that allowed to confirm the genotype-phenotype relationship and to further unravel the molecular causes of the disease and the mechanisms underlying primary haemostasis. More recently, interest has been growing in the effects of the more common polymorphisms that are found in the platelet glycoproteins as possible risk factors for thrombotic disorders. The new era of platelet genomics and proteomics will increase our knowledge on platelet disorders that will improve their diagnosis, but also will provide basis for new antithrombotic therapies.

Cytotoxic activity of Bacillus anthracis protective antigen observed in a macrophage cell line overexpressing ANTXR1.

Anthrax toxin protective antigen (PA) binds cell surface receptors (e.g. ANTXR1,2), forms heptameric pores, and translocates lethal factor (LF) or oedema factor (OF) into the cytoplasm of mammalian cells. In the current study, we sought to determine how receptor levels influence these events, by examining PA heptamer stability and related processes in macrophages that overexpress ANTXR1 (RAW 264.7ANTXR1). In these experiments, PA-oligomers demonstrated an extended half-life in RAW 264.7ANTXR1 macrophages, with SDS-resistant heptamers detected up to 10 h following treatment, while levels of PA-oligomers declined within 3 h in control cells. RAW 264.7ANTXR1 macrophages were also more sensitive to lethal toxin, a combination of PA and LF. Surprisingly, we found that PA alone was cytotoxic to RAW 264.7ANTXR1 cells. Further analysis found that PA cytotoxicity required direct interaction with ANTXR1, oligomerization, channel formation, endosomal acidification, and was independent of the ANTXR1 cytoplasmic tail. PA intoxication of RAW 264.7ANTXR1 macrophages resulted in caspase-3 activation, with corresponding DNA fragmentation and proteolytic cleavage of poly-ADP-ribose polymerase, as well as activation of Bid, suggesting cell death occurred via apoptosis. Overall, results from the current study suggest that receptor levels dictate the extent of PA oligomer stability, and shifts in this normal process can lead to cell death via apoptosis in the absence of toxin catalytic subunits.

Bacillus anthracis oedema toxin as a cause of tissue necrosis and cell type-specific cytotoxicity.

Oedema factor (OF) and protective antigen (PA) are secreted by Bacillus anthracis, and their binary combination yields oedema toxin (OT). Following PA-mediated delivery to the cytosol, OF functions as an adenylate cyclase generating high levels of cAMP. To assess OT as a possible cause of tissue damage and cell death, a novel approach was developed, which utilized a developing zebrafish embryo model to study toxin activity. Zebrafish embryos incubated with OT exhibited marked necrosis of the liver, cranium and gastrointestinal tract, as well as reduced swim bladder inflation. The OT-treated embryos survived after all stages of development but succumbed to the toxin within 7 days. Additional analysis of specific cell lines, including macrophage and non-macrophage, showed OT-induced cell death is cell type-specific. There was no discernible correlation between levels of OF-generated cAMP and cell death. Depending on the type of cell analysed, cell death could be detected in low levels of cAMP, and, conversely, cell survival was observed in one cell line in which high levels of cAMP were found following treatment with OT. Collectively, these data suggest OT is cytotoxic in a cell-dependent manner and may contribute to disease through direct cell killing leading to tissue necrosis.

Toxin-induced resistance in Bacillus anthracis lethal toxin-treated macrophages.

In the current study, we show that macrophages adaptively resist anthrax lethal toxin (LT) through a toxin-activated process termed toxin-induced resistance (TIR). TIR was triggered by pretreatment of RAW 264.7 or J774A.1 macrophages with a low dose of LT for at least 6 h, which resulted in resistance to high doses of LT for 96 h. Activation of TIR required functional toxin, because LT subunits, mutants, and heat-inactivated toxin were unable to trigger resistance. TIR macrophages were not altered in toxin receptor levels or cell cycle profiles. Treatment of TIR macrophages with high doses of LT resulted in a sustained decline in full-length mitogen-activated protein kinase kinase 2, a known target of lethal factor, and a marked reduction in diphosphorylated extracellular response kinases 1,2 for 24 h. However, despite the sustained loss of full-length mitogen-activated protein kinase kinase 2, by 48 h, TIR macrophages regained diphosphorylated extracellular response kinases 1,2, suggesting an adaptation led to recovery of this signaling pathway. TIR macrophages were also able to maintain normal levels of ubiquitinylated proteins, whereas sensitive cells show a rapid reduction in ubiquitin-modified proteins before cell death, indicating a possible alteration in proteasome activity contributed to resistance. These results provide a paradigm for toxin-cell interactions and suggest macrophages are capable of adapting to and tolerating toxic doses of LT.

Decreased glycogen synthase kinase 3-beta levels and related physiological changes in Bacillus anthracis lethal toxin-treated macrophages.

The lethal factor (LF) component of Bacillus anthracis lethal toxin (LeTx) cleaves mitogen activated protein kinase kinases (MAPKKs) in a variety of different cell types, yet only macrophages are rapidly killed by this toxin. The reason for this selective killing is unclear, but suggests other factors may also be involved in LeTx intoxication. In the current study, DNA membrane arrays were used to identify broad changes in macrophage physiology after treatment with LeTx. Expression of genes regulated by MAPKK activity did not change significantly, yet a series of genes under glycogen synthase kinase-3-beta (GSK-3beta) regulation changed expression following LeTx treatment. Correlating with these transcriptional changes GSK-3beta was found to be below detectable levels in toxin-treated cells and an inhibitor of GSK-3beta, LiCl, sensitized resistant IC-21 macrophages to LeTx. In addition, zebrafish embryos treated with LeTx showed signs of delayed pigmentation and cardiac hypertrophy; both processes are subject to regulation by GSK-3beta. A putative compensatory response to loss of GSK-3beta was indicated by differential expression of three motor proteins following toxin treatment and Kif1C, a motor protein involved in sensitivity to LeTx, increased expression in toxin-sensitive cells yet decreased in resistant cells following toxin treatment. Differential expression of microtubule-associating proteins and a decrease in the level of cellular tubulin were detected in LeTx-treated cells, both of which can result from loss of GSK-3beta activity. These data provide new information on LeTx's overall influence on macrophage physiology and suggest loss of GSK-3beta contributes to cytotoxicity.