Exposure of endogenous phosphatidylserine at the outer surface of stimulated platelet is reversed by restoration of aminophospholipid translocase activity

Department of Biochemistry, University of Limburg, Maastricht, The Netherlands.
Biochemistry (Impact Factor: 3.02). 03/1989; 28(6):2382-7. DOI: 10.1021/bi00432a007
Source: PubMed


Phosphatidylserine (PS) in the plasma membrane of nonactivated human platelets is almost entirely located on the cytoplasmic side. Stimulation of platelets with the Ca2+ ionophore A23187 or combined action of collagen plus thrombin results in a rapid loss of the asymmetric distribution of PS. Also, treatment with the sulfhydryl-reactive compounds diamide and pyridyldithioethylamine (PDA) causes exposure of PS at the platelet outer surface. PS exposure is sensitively measured as the catalytic potential of platelets to enhance the rate of thrombin formation by the enzyme complex factor Xa-factor Va, since this reaction is essentially dependent on the presence of a PS-containing lipid surface. In this paper we demonstrate that endogenous PS, previously exposed at the outer surface during cell activation or sulfhydryl oxidation, can be translocated back to the cytoplasmic leaflet of the membrane by addition of dithiothreitol (DTT) but not by nonpermeable reducing agents like reduced glutathione. Treatment of platelets with trypsin or chymotrypsin, prior to addition of DTT, inhibits the inward transport of exposed PS. Moreover, severe depletion of metabolic ATP, as obtained by platelet stimulation with A23187 in the presence of metabolic inhibitors, though not inhibiting PS exposure at the outer surface, blocks the translocation of endogenous PS to the internal leaflet of the plasma membrane. These results strongly indicate the involvement of a membrane protein in the inward transport of endogenous PS. Recently, an aminophospholipid-specific translocase in the platelet membrane was postulated on the basis of the inward transport of exogenously added PS (analogues) [Sune, A., Bette-Bobillo, P., Bienvenue, A., Fellmann, P., & Devaux, P.F. (1987) Biochemistry 26, 2972-2978].(ABSTRACT TRUNCATED AT 250 WORDS)

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    • "Recently, some reports have described a membrane lipid rearrangement occurring in differently triggered apoptosis , and clearly shown by FITC-annexin V labelling. This stain is highly specific to PS, which can therefore be clearly localised when it is exposed on the external membrane surface following rearrangement of the lipid bilayer (Bevers et al. 1989; Boersma et al. 1996; Fadok et al. 1992; Koopman et al. 1994; Verhoven et al. 1995; Vermes et al. 1995). "
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    ABSTRACT:  The behaviour of plasma membrane was studied in UV-treated cells to investigate its involvement in apoptosis. It was studied in HL60 cells, in which DNA oligonucleosomic cleavage occurs, and in Molt-4 cells, which are characterised by a different fragmentation pattern. During the early stages of apoptosis, a membrane lipid rearrangement occurs, which involves phosphatidylserine translocation from the inner to the outer leaflet. This molecular alteration was investigated by annexin V-FITC binding, analysed by flow cytometry and confocal microscopy. It was correlated with transmission electron microscopy, subdiploid peak appearance and DNA fragmentation. Our data indicate that the plasma membrane represents an early apoptotic target, even if its alterations are not detectable by ultrastructural analysis, which indicates its good preservation until late apoptotic stages. In addition, the study of apoptotic cells with absent or inactivated endonuclease demonstrates the independence of this membrane mechanism from nuclear activity.
    Histochemie 09/1998; 110(5):467-476. DOI:10.1007/s004180050308 · 3.05 Impact Factor
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    • "Nevertheless, the mechanism by which they expose aminophospholipids is unknown. Agents such as N-ethyl maleimide (NEM) and pyridyl-dithioethylamine (Morrot et al, 1989; Bevers et al, 1989) expose aminophospholipids in platelets most probably through their reaction with free sulphydryl groups in critical enzymes including the aminophospholipid translocase and calpain. Ca 2þ -ATPases of the sarcoplasmic reticulum also contain several classes of thiols (Yamada & Ikemoto, 1978). "
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    ABSTRACT: The Scott syndrome is a rare inherited haemorrhagic disorder characterized by the inability of blood cells to expose aminophospholipids and to shed microparticles. We have had the opportunity to study a recently reported French patient with this syndrome and have confirmed by means of a fluorescence assay for transbilayer lipid movement a reduced aminophospholipid exposure when platelets were stimulated with the calcium-ionophore ionomycin, in spite of a normal elevation of intracellular Ca2+. Secretion and calpain activation were also shown to be normal. Significantly, the level of phosphotyrosine-labelled proteins in platelets treated with thrombin or a thrombin + collagen mixture and in particular the phosphorylation of a 40 kD band were severely reduced. Furthermore, inhibition of thiol-containing enzymes. including tyrosine-phosphatases, by N-ethyl maleimide did not lead to aminophospholipid exposure in the patient's platelets, in spite of increased tyrosine protein phosphorylation. In contrast, amphiphilic membrane drugs such as tetracaine and propranolol induced both surface aminophospholipid exposure in Scott platelets and the shedding of microparticles, thereby showing that membrane perturbation can lead to loss of phospholipid asymmetry in this syndrome. Our results provide the first insight that the lack of expression of procoagulant phospholipids and microparticle formation in Scott syndrome platelets is associated with a defect of intracellular signalling.
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    ABSTRACT: Blood coagulation is initiated following injury to the blood vessel wall through the exposure of tissue factor, which mediates the activation of circulating factor VII. Activated factor VIIa forms a complex with tissue factor and triggers the subsequent steps in the activation of the coagulation system, ultimately culminating in the conversion of prothrombin to thrombin (1–5). The platelet membrane surface is crucial for this process, providing a milieu, referred to as the prothrombinase complex, for efficient assembly of the coagulation complexes and localization of the procoagulant response. Indeed, recent data have even implicated platelets in the initiation of coagulation as they acquire circulating forms of tissue factor, generated from other cells, on their surface (6,7). Thrombin mediates fibrin formation and plays a critical role in the inflammatory responses (8); thus, the regulation of prothrombin activation by the platelet membrane has broad physiological and pathophysiological implications.
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