Platelet Ca2+ Homeostasis: Na+-Ca2+ Exchange in Plasma Membrane Vesicles

SummaryA vesicular plasma membrane-enriched fraction obtained from human platelets exhibited 45Ca2+ uptake in exchange for intravesicular Na+. The rate of Ca2+ uptake was linear up to 4 sec. The apparent Km for Ca2+ was 22 pM and the Vmax 280 pmol/mg/ sec. Ca2+ efflux from Ca2+ loaded vesicles was obtained upon dilution into a NaCl but not a KC1 medium. The extent of Ca2+uptake was monotonically increased as the pH increased from 6 to 9. Na+-Ca2+ exchange was shown to be electrogenic. Ca2+ uptake was distinguished from binding by the induction of Ca2+ release after A23187 addition. These findings support a role for Na+-Ca2+ exchange in human platelet Ca2+ transport.

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Isolation of InsP4 and InsP6 binding proteins from human platelets: InsP4 promotes Ca2+ efflux from inside-out plasma membrane vesicles containing 104 kDa GAP1IP4BP protein

Biochemical Journal ◽

10.1042/bj3151027 ◽

1996 ◽

Vol 315 (3) ◽

pp. 1027-1034 ◽

Cited By ~ 21

Author(s):

Flavia O'ROURKE ◽

Eileen MATTHEWS ◽

Maurice B. FEINSTEIN

Keyword(s):

Plasma Membrane ◽

Calcium Oxalate ◽

Binding Sites ◽

Amino Acid Content ◽

Membrane Vesicles ◽

Human Platelets ◽

Binding Activity ◽

Plasma Membrane Vesicles ◽

Internal Peptide ◽

Inside Out

A low-density membrane fraction from human platelets contained the plasma membrane marker glycoprotein Ib (GpIb) and selective binding sites for InsP4 and InsP6. It was separated from the bulk of InsP3-receptor-containing membranes, but was heterogeneous, probably also containing surface-connected canalicular system and some lighter elements of the internal dense tubule system. After loading with calcium oxalate and re-centrifugation on Percoll gradients, this mixed fraction was subfractionated into light membranes containing all of the GpIb, high-affinity InsP4 binding sites (KD = 18 nM) and phosphate-stimulated Ca2+ transport activity. InsP4 (EC50 0.6 μM), but not InsP3 or InsP6, released up to 35% of the accumulated Ca2+ from these vesicles, which were shown to be inside-out plasma membrane vesicles by a biotinylation labelling technique and selective removal of right-side-out plasma membrane vesicles with streptavidin–agarose. Most of the InsP4, and all of the InsP6, binding was present in the much denser calcium oxalate-loaded subfractions, which were free of GpIb. InsP6 binding activity was chromatographically purified as a 116 kDa protein (KD for InsP6 = 5.9 nM), with an amino acid content and two internal peptide sequences identical to those of 116 kDa vinculin. A 104 kDa InsP4 binding protein (KD for InsP4 = 12 nM), probably identical to GAP1IP4BP described by Cullen, Hsuan, Truong, Letcher, Jackson, Dawson and Irvine [(1995) Nature (London) 376, 527–530], was also isolated. This InsP4 receptor may mediate Ca2+ influx in platelets that occurs subsequent to receptor-stimulated production of InsP3 and unloading of internal Ca2+ stores.

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Loss of phospholipid asymmetry in dilauroylphosphatidylcholine induced plasma membrane vesicles from human platelets

Biochimica et Biophysica Acta (BBA) - Biomembranes ◽

10.1016/0005-2736(87)90169-6 ◽

1987 ◽

Vol 903 (1) ◽

pp. 197-205 ◽

Cited By ~ 9

Author(s):

Edouard M. Bevers ◽

Peter F.J. Verhallen ◽

Willie M.A. Linskens ◽

Paul Comfurius ◽

Robert F.A. Zwaal

Keyword(s):

Plasma Membrane ◽

Membrane Vesicles ◽

Human Platelets ◽

Plasma Membrane Vesicles ◽

Phospholipid Asymmetry

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Isolation of plasma membrane vesicles of human platelets by affinity chromatography

Thrombosis Research ◽

10.1016/0049-3848(84)90040-9 ◽

1981 ◽

Vol 21 (1-2) ◽

pp. 129-135 ◽

Cited By ~ 3

Author(s):

J. Kambayashi ◽

M. Sakon ◽

H. Ohno ◽

G. Kòsaki

Keyword(s):

Plasma Membrane ◽

Affinity Chromatography ◽

Membrane Vesicles ◽

Human Platelets ◽

Plasma Membrane Vesicles

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Platelet glutathione transport: characteristics and evidence for regulation by intraplatelet thiol status

Biochemistry and Cell Biology ◽

10.1139/o95-019 ◽

1995 ◽

Vol 73 (3-4) ◽

pp. 155-162 ◽

Cited By ~ 8

Author(s):

Daniel J. Sexton ◽

Bulent Mutus

Keyword(s):

Plasma Membrane ◽

Membrane Vesicles ◽

Membrane Depolarization ◽

Human Platelets ◽

Plasma Membrane Vesicles ◽

Induced Membrane ◽

Glutathione Transport

The present study demonstrates the carrier-mediated uptake of intact glutathione (GSH) by human platelets. Platelet GSH uptake was characterized as being Na+independent and saturable. The KM, apparentand Vmax, apparentfor GSH uptake in platelet plasma membrane vesicles were 28.0 ± 8.4 μM and 263.5 ± 28.5 pmol/min per mg protein, respectively. The transport was inhibited by GSH analogs and enhanced by KCl-induced membrane depolarization. GSH transport may be regulated by the intracellular thiol status, since the depletion of intraplatelet GSH with 100 μM 1-chloro-2,4-dinitrobenzene (CDNB) increased GSH uptake by ~40%. The KM, apparentand Vmax, apparentfor GSH uptake in intact platelets changed from 99.5 ± 15 μM and 42 ± 7.5 pmol/min per 109platelets, respectively, to 33.7 ± 6.7 μM and 21.5 ± 6.9 pmol/min per 109platelets, respectively, on reducing intraplatelet GSH with 100 μM CDNB.Key words: glutathione, platelets, transport.

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