Association with actin mediates the EGTA-resistant binding of cytosolic phospholipase A 2-α to the plasma membrane of activated platelets
ABSTRACT The association of cytosolic phospholipase A2-α (cPLA2α) with intracellular membranes is central to the generation of free arachidonic acid and thromboxane A2 in activated platelets. Despite this, the site and nature of this membrane association has not been fully characterised upon platelet activation. High resolution imaging showed that cPLA2α was distributed in a partly structured manner throughout the resting platelet. Upon glass activation or thrombin stimulation, cPLA2α relocated to a peripheral region corresponding to the platelet plasma membrane. Upon thrombin stimulation of platelets a major pool of cPLA2α was associated with the plasma membrane in an EGTA-resistant manner. EGTA-resistant membrane binding was abolished upon de-polymerisation of actin filaments by DNase I and furthermore, cPLA2α co-immunoprecipitated with actin upon thrombin stimulation of platelets. Immunofluorescence microscopy studies revealed that, upon platelet activation, cPLA2α and actin co-localised at the plasma membrane. Thus we have identified a novel mechanism for the interaction of cPLA2α with its membrane substrate via interaction with actin.
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ABSTRACT: Thromboxane A2 (TXA2) is a labile metabolite of arachidonic acid that has potent biological effects. Its actions are mediated by G protein-coupled thromboxane-prostanoid (TP) receptors. TP receptors have been implicated in the pathogenesis of cardiovascular diseases. To investigate the physiological functions of TP receptors, we generated TP receptor-deficient mice by gene targeting. Tp-/- animals reproduce and survive in expected numbers, and their major organ systems are normal. Thromboxane agonist binding cannot be detected in tissues from Tp-/- mice. Bleeding times are prolonged in Tp-/- mice and their platelets do not aggregate after exposure to TXA2 agonists. Aggregation responses after collagen stimulation are also delayed, although ADP-stimulated aggregation is normal. Infusion of the TP receptor agonist U-46619 causes transient increases in blood pressure followed by cardiovascular collapse in wild-type mice, but U-46619 caused no hemodynamic effect in Tp-/- mice. Tp-/- mice are also resistant to arachidonic acid-induced shock, although arachidonic acid signifi-cantly reduced blood pressure in Tp-/- mice. In summary, Tp-/- mice have a mild bleeding disorder and altered vascular responses to TXA2 and arachidonic acid. Our studies suggest that most of the recognized functions of TXA2 are mediated by the single known Tp gene locus.Journal of Clinical Investigation 01/1999; 102(11):1994-2001. · 12.81 Impact Factor
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ABSTRACT: Cytosolic phospholipase A2 is a Ca2+-dependent enzyme that acts on membrane phospholipids to release arachidonic acid, which in platelets is converted to thromboxane A2. Annexin V is a Ca2+-dependent, phospholipid-binding protein, which is proposed to regulate inflammation by inhibiting cytosolic phospholipase A2. Here, we have studied the association of cytosolic phospholipase A2 and annexin V with platelet membranes after thrombin stimulation. In a time-dependent manner, an exact correlation was found between the membrane association of cytosolic phospholipase A2 and annexin V. Calcium from the intracellular stores was sufficient for the relocation of intracellular annexin V and cytosolic phospholipase A2 to platelet membranes. Activation in the presence of arginyl-glycyl-aspartyl-serine (RGDS), which inhibits binding of fibrinogen to its adhesive ligand, does not alter the amount of cytosolic phospholipase A2 or annexin V that binds to membranes. When activation-induced actin polymerisation was prevented by cytochalasin E, the recovery of both annexin V and cytosolic phospholipase A2 remained unchanged. However, complete depolymerisation of the cytoskeleton with DNase I almost abolished the association of cytosolic phospholipase A2 with the membranes, and it completely abolished the relocation of annexin V to platelet membranes. Finally, we show that cytosolic phospholipase A2 can be specifically purified from platelet membranes by affinity chromatography on GST-annexin V and that immunoprecipitation using antibodies against cytosolic phospholipase A2 copurify annexin V and cytosolic phospholipase A2 from activated platelets. These findings suggest that following platelet activation with thrombin, both cytosolic phospholipase A2 and annexin V, relocate to platelet membranes where they interact. An intact cytoskeleton seems to be a prerequisite for the interaction of cytosolic phospholipase A2 and annexin V with platelet membranes. The incorporation of cytosolic phospholipase A2 into the membrane fraction of thrombin-activated platelets parallels that of annexin V, which suggests an interaction between the two proteins.Thrombosis Research 04/2000; 97(6):421-429. · 3.13 Impact Factor
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ABSTRACT: Modulation of cytosolic phospholipase A(2) (cPLA(2)) activity by sphingomyelin (SPH), ceramide (Cer), and cholesterol (Chol) was investigated in CHO-2B cells activated by the calcium ionophore A23187 and epinephrine. Chol depletion of CHO-2B cells by treatment with methyl-beta-cyclodextrin (5 mm) resulted in the inhibition of the release of arachidonic acid whereas the restoration of the level by Chol-loaded cyclodextrin relieved inhibition. Conversion of CHO-2B cellular SPH to Cer by Staphylococcus aureus sphingomyelinase enhanced endogenous cPLA(2) activation as well as uptake by cells of C2- and C6-ceramide analogs. These results were confirmed in vitro with purified human recombinant cPLA(2) acting on a model phospholipid substrate. The enzyme activity was inhibited by SPH but reactivated by Cer as well as by Chol added to glycerophospholipid liposomal substrates containing SPH. The results of this study, which combine in situ and in vivo experimental approaches, indicate that membrane microdomains enriched in SPH and Chol play a role in the modulation of the activity of cPLA2 and in arachidonic acid-derived mediator production.The Journal of Lipid Research 11/2000; 41(10):1680-8. · 4.39 Impact Factor