Abstract
It is unknown whether the endocytosis-independent transfer of phospholipids from lipoproteins to platelets is regulated by platelet agonists such as thrombin. The movements of the choline phospholipids phosphatidylcholine and sphingomyelin (labeled with either 14C or the fluorescent pyrenedecanoic acid) between low density lipoproteins and platelets were unaffected by thrombin (0.5 unit/ml). In contrast, thrombin accelerated the import of diacyl phosphatidylethanolamine (PE) and alkenylacyl phosphatidylethanolamine into platelets by about 4-fold. Similarly, thrombin receptor-activating peptide (15 microM), collagen (10 microgram/ml), and ADP (10 microM) enhanced PE uptake. High density lipoprotein particles and egg phosphatidylcholine vesicles were also donors for stimulation of platelet PE import. Part of the [14C]arachidonic acid-labeled PE transferred from low density lipoprotein to platelets activated by thrombin and collagen was metabolized to 14C-eicosanoids. Inhibitors of protein kinase C partially prevented thrombin-induced [14C]PE uptake, while direct activators of protein kinase C increased incorporation of [14C]PE into platelets. Proteinaceous factor(s) recovered in the extracellular medium from ADP- and thrombin-activated platelet suspensions were found to accelerate the transfer of pyrenedecanoic acid-labeled PE between donor and acceptor lipid vesicles. The stimulation of import of ethanolamine phospholipids led to a 2-fold enhancement of the prothrombinase activity of thrombin-activated platelets. Our study demonstrates that physiological platelet stimuli increase specifically the transfer of ethanolamine phospholipids from lipoproteins to platelets through a secretion-dependent mechanism. This might contribute to the increase of procoagulant activity of stimulated platelets.
Highlights
The processes of phospholipid transfer between different plasma lipoproteins [1], between the two leaflets of biological membranes [2], as well as between different intracellular membranes [3] have been elucidated in considerable detail in recent years
In order to prevent platelet aggregation, which might lead to trapping of labeled lipoproteins, platelets were pretreated for 20 min with 2 mM EGTA before adding thrombin
We observed in the present study that the platelet stimuli thrombin, collagen, and ADP enhance the import of ethanolamine phospholipids into platelets
Summary
Materials—1-Palmitoyl-2-[14C]arachidonoyl-sn-glycero-3-phosphorylcholine (14C-20:4-PC), 1-acyl-2-[14C]arachidonoyl-sn-glycero-3-phosphorylethanolamine (14C-20:4-PE), its [14C]linoleic acid analogue, and [N-methyl-14C]sphingomyelin ([14C]SM) were obtained from Amersham (Braunschweig, Germany) or from NEN DuPont (Homburg). 1-Palmitoyl-2-pyrenedecanoyl-sn-3-glycerophosphorylcholine (py-PC), 1-palmitoyl-2-pyrenedecanoyl-sn-3-glycerophosphorylethanolamine (py-PE), and (N-pyrenedecanoyl)-sphingomyelin (py-SM) were from Sigma (Deisenhofen, Germany) or from Molecular Probes (Eugene, OR). 1-Alkenyl-2-pyrenedecanoyl-sn-3-glycerophosphorylcholine (py-PPC) and 1-alkenyl-2-pyrenedecanoyl-sn-3-glycerophosphorylethanolamine (pyPPE) were synthesized as described [13]. 125I-LDL (6 ϫ 108 cpm/mg LDL protein; prepared by the IODOGEN method) was a kind gift of Dr E. For the preparation of phospholipid vesicles, 250 g of egg phosphatidylcholine and 5 g of py-PC, py-PPC, py-PE, py-PPE, or py-SM were dissolved in 30 l of ethanol and thereafter slowly injected under argon into 2 ml of a buffer containing (in mM): 138 NaCl, 3 KCl, 1 MgCl2, 15 Hepes, 9 citrate, 5 EDTA, 5 glucose (pH 7.4, “incubation buffer”). In some cases, following incubation of platelets with pyrene-labeled donors, fluorescence intensities were determined after separation of donors and acceptors by centrifugation (off-line experiments). Platelets (5 ϫ 106/0.5 ml) were incubated without or with the indicated concentrations of thrombin and collagen plus either LDL or phospholipid vesicles for 5 min at 37 °C in a buffer composed of (in mM): 145 NaCl, 10 Hepes, 5 KCl, 1 MgCl2, 5 glucose (pH 7.4, “coagulation buffer”).
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