Pathophysiologic Implications of Membrane Phospholipid Asymmetry in Blood Cells

Cardiovascular Research Institute Maastricht, Maastricht University, The Netherlands.
Blood (Impact Factor: 10.45). 03/1997; 89(4):1121-32.
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Available from: R.F.A. Zwaal,
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    • "Under normal cellular homeostasis, phosphatidylserine (PS) is an anionic membrane phospholipid preferentially located on the inner leaflet of the plasma membrane. This asymmetry is maintained by ATP-dependent aminophospholipid translocases that catalyze the transfer of aminophospholipids from the external to the internal plasma membrane [1] [2]. In certain physiologic states, such as apoptosis and cell activation, PS asymmetry is disrupted, resulting in exposure of PS on the external membrane leaflet [3-6]. "
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    ABSTRACT: Bavituximab is a chimeric monoclonal antibody with immune modulating and tumor-associated vascular disrupting properties demonstrated in models of non-small cell lung cancer (NSCLC). The molecular target of Bavituximab, phosphatidylserine (PS), is exposed on the outer leaflet of the membrane bi-layer of malignant vascular endothelial cells and tumor cells to a greater extent than on normal tissues. We evaluated the tumor-targeting properties of Bavituximab for imaging of NSCLC xenografts when radiolabeled with (111)In through conjugation with a bifunctional chelating agent, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). In vitro binding of (111)In-DOTA-Bavituximab to PS was determined by enzyme-linked immunosorbent assay (ELISA). Biodistribution of (111)In-DOTA-Bavituximab was conducted in normal rats, which provided data for dosimetry calculation. Single-photon emission computed tomography/computed tomography (SPECT/CT) imaging was performed in athymic nude rats bearing A549 NSCLC xenografts. At the molar conjugation ratio of 0.54 DOTA per Bavituximab, the PS binding affinity of (111)In-DOTA-Bavituximab was comparable to that of unmodified Bavituximab. Based on the quantitative SPECT/CT imaging data analysis, (111)In-DOTA-Bavituximab demonstrated tumor-specific uptake as measured by the tumor-tomuscle ratio, which peaked at 5.2 at 72 hr post-injection. In contrast, the control antibody only presented a contrast of 1.2 at the same time point.These findings may underlie the diagnostic efficacy and relative low rates of systemic vascular and immune-related toxicities of this immunoconjugate. Future applications of (111)In-DOTA-bavituximab may include prediction of efficacy, indication of tumor immunologic status, or characterization of radiographic findings.
    American Journal of Nuclear Medicine and Molecular Imaging 11/2015; 5(5):493-503. · 3.25 Impact Factor
    • "Phosphatidylserine together with the major part of phosphatidylethanolamine (PE) assembles the inner leaflet of eukaryotic non-cancerous plasma membranes [21]. This lipid asymmetry of the two membrane leaflets is well documented [22] [23] and is maintained by an ATPdependent aminophospholipid translocase [24]. The loss of membrane asymmetry was shown for several cancer types, including metastases, promising to be a general marker of cancer cells and target for therapy by peptides [13,15–18,20,25,26]. "
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    ABSTRACT: Host defense-derived peptides have emerged as a novel strategy for the development of alternative anticancer therapies. In this study we report on characteristic features of human lactoferricin (hLFcin) derivatives which facilitate specific killing of cancer cells of melanoma, glioblastoma and rhabdomyosarcoma compared with non-specific derivatives and the synthetic peptide RW-AH. Changes in amino acid sequence of hLFcin providing 9-11 amino acids stretched derivatives LF11-316, -318 and -322 only yielded low antitumor activity. However, the addition of the repeat (di-peptide) and the retro repeat (di-retro-peptide) sequences highly improved cancer cell toxicity up to 100% at 20 μM peptide concentration. Compared to the complete parent sequence hLFcin the derivatives showed toxicity on the melanoma cell line A375 increased by 10-fold and on the glioblastoma cell line U-87mg by 2-3-fold. Reduced killing velocity, apoptotic blebbing, activation of caspase 3/7 and formation of apoptotic DNA fragments proved that the active and cancer selective peptides, e.g. R-DIM-P-LF11-322, trigger apoptosis, whereas highly active, though non selective peptides, such as DIM-LF11-318 and RW-AH seem to kill rapidly via necrosis inducing membrane lyses. Structural studies revealed specific toxicity on cancer cells by peptide derivatives with loop structures, whereas non-specific peptides comprised α-helical structures without loop. Model studies with the cancer membrane mimic phosphatidylserine (PS) gave strong evidence that PS only exposed by cancer cells is an important target for specific hLFcin derivatives. Other negatively charged membrane exposed molecules as sialic acid, heparan and chondroitin sulfate were shown to have minor impact on peptide activity. Copyright © 2015. Published by Elsevier B.V.
    Biochimica et Biophysica Acta 07/2015; 1848(11). DOI:10.1016/j.bbamem.2015.07.018 · 4.66 Impact Factor
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    • "Finally, Ca 2+ can inhibit the phospholipid flippase directly, while promoting scramblase activity (Balasubramanian et al, 2007;Zwaal & Schroit, 1997). An increased concentration of intracellular free Ca 2+ could therefore lead to a higher scramblase activity, relative to the flippase, resulting in PS exposure. "
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    DESCRIPTION: Patients with the rare, X-linked, mitochondrial disorder Barth syndrome (BTHS) suffer from moderate to severe neutropenia amongst other symptoms. BTHS is caused by mutations in the TAZ gene, encoding tafazzin, which is involved in cardiolipin metabolism, a phospholipid restricted to the mitochondrial inner membrane. It has previously been reported that neutrophils from BTHS patients avidly bind annexin V, indicative of phosphatidyl serine (PS) exposure -a well-described ‘eat me’ signal for apoptotic cells- in the absence of apoptosis. PS exposure is normally actively prevented by a fast acting lipid transporter called flippase. In this study, we addressed the question why only neutrophils, and not other immune cells, are affected in BTHS patients. We found that ATP levels in BTHS neutrophils were normal even though their mitochondria were consistently defective. In addition, we demonstrate a linear inverse correlation between the absolute neutrophil cell count in BTHS patients and their level of annexin-V binding. Finally, we uncover a potential link between reduced mitochondrial membrane potential observed in BTHS cells and flippase inhibition by Ca2+ in neutrophils, leading to PS exposure. We hypothesize that this mechanism may be responsible for increased neutrophil clearance and neutropenia observed in BTHS patients.
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