Phospholipase D in human blood platelets
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We have studied phospholipase D (PLD) in human blood platelets. This enzyme hydrolyzes phosphatidylcholine to phosphatidic acid (PA) and choline, where PA is considered to be the main effector of PLDs function in cells. PA is reported to function as a second messenger, involved in membrane protein recruitment and membrane fusion processes, and PLD is proposed to play a role in signalling, intracellular transport and cytoskeletal rearrangements in cells. The role and regulation of PLD in platelets are largely unknown. In this study we report that both isoforms of PLD, PLD1 and 2, are present in platelets. In resting platelets the two isozymes were localized all over the cells and upon addition of the platelet agonist thrombin they rapidly translocated to the membrane area. We showed that thrombininduced PLD activity was enhanced by extracellular Ca2+ and autocrine stimulation, notably by ADP and binding of fibrinogen to its receptor. The thrombin-induced translocation was independent of Ca2+, autocrine stimulation or PA from the PLD reaction, thus a primary thrombin effect. We found that the platelet antagonist PGE1 was able to induce a modest PLD activity at the same time as it inhibited PLD activation by thrombin. Further investigations using forskolin, inhibitors and specific activators of protein kinase A (PKA) and G, indicated that thrombin-induced PLD activity was inhibited by PKA. We observed that PLD1 and PLD2 had different regulation mechanisms in platelets as PKA/forskolin only inhibited PLD1 translocation by thrombin and also as phorbol 12–myristate 13-acetate (PMA), a direct activator of protein kinase C (PKC) only was able to induce PLD1 translocation. We wanted to study possible interactions between PLD and PKC isoenzymes by immunoprecipitation as previously demonstrated in C3H10T1/2 fibroblasts. We observed co-precipitation of both PLD1 and 2 with all PKC isoenzymes investigated in both stimulated and unstimulated platelets. PKCα showed a constitutive association with both PLD1 and 2 independent of the agents added (thrombin, PMA, forskolin or PGE1), while the association between PLDs and PKC βI, βII and δ varied with the different conditions. PLD1 and PLD2 associated differently with the PKC isoenzymes, again indicating different regulation mechanisms. We also report that PLD1 and 2 associated with PLCβII, which is believed to be upstream of PKC in the platelet activation pathway mediated by thrombin. Our findings that PLD1 and 2 associated with different PKC isoforms believed to be involved in distinct different mechanisms in platelets, indicate different roles for the PLD isozymes. PLD in platelets is thought so far to be implied in aggregation and secretion; we suggest in this work by correlation-studies, however, that PLD might be involved in lysosomal secretion and F-actin formation.
Has partsPaper I: Platelets 19(3), Vorland, M.; Holmsen, H., Phospholipase D in human platelets: Presence of isoenzymes and participation of autocrine stimulation during thrombin activation, pp. 211-224. Copyright 2008 Informa UK Ltd. Published by Taylor & Francis. Full text not available in BORA due to publisher restrictions. The published version is available at: http://dx.doi.org/10.1080/09537100701777329
Paper II: Platelets 19(4), Vorland, M.; Holmsen, H., Phospholipase D activity in human platelets is inhibited by protein kinase A, involving inhibition of phospholipase D1 translocation, pp. 300-307. Copyright 2008 Informa UK Ltd. Published by Taylor & Francis. Full text not available in BORA due to publisher restrictions. The published version is available at: http://dx.doi.org/10.1080/09537100801910838
Paper III: Vorland, M.; Holmsen, H., 2008, Phospholipase D in human platelets. Involvement of PKC isoenzymes and PLCβII. Full text not available in BORA.
Paper IV: Platelets 19(8), Vorland, M.; Thorsen, V. A. T.; Holmsen, H., Phospholipase D in platelets and other cells, pp. 582-594. Copyright 2008 Informa UK Ltd. Published by Taylor & Francis. Full text not available in BORA due to publisher restrictions. The published version is available at: http://dx.doi.org/10.1080/09537100802454992