ABSTRACT
BACKGROUND: Blood platelets are small anucleated cell fragments generated from bone marrow megakaryocytes (MKs) by a cytoskeleton-driven process. Thereby, mature MKs form long cytoplasmic protrusions (pro-platelets), which extend into the sinusoids within the bone marrow and finally release platelets. Podosomes are F-actin rich matrix contacts that have been suggested to play an important role in cell migration, but also in pro-platelet formation by MKs. Phospholipase D (PLD) has been proposed to contribute to the regulation of actin dynamics through the local generation of phosphatidic acid but its role in platelet formation is unknown. OBJECTIVE: We sought to investigate the significance of PLD in MK podosome formation and thrombocytopoiesis. METHODS: Podosome formation, spreading and ultra-structure of PLD single- and double-deficient MKs were analyzed using confocal and transmission electron microscopy. RESULTS: Phospholipase D-deficient MKs displayed a highly altered ultra-structure in vivo and abnormal actin rearrangement, with almost abolished formation of podosomes upon spreading on collagen I in vitro. However, MK endomitosis and platelet production were not altered by PLD deficiency. CONCLUSION: Together, our findings point to a specific function of PLD in actin dynamics as well as podosome formation and size determination in MKs on a collagen I matrix. The normal platelet number in PLD-deficient mice, however, suggests the existence of compensatory mechanisms in vivo that overcome the defective podosome formation observed in vitro.
Subject(s)
Collagen Type I/physiology , Cytoskeleton/metabolism , Megakaryocytes/metabolism , Phospholipase D/metabolism , Animals , Mice , Mice, Knockout , Microscopy, Electron, Transmission , Phospholipase D/geneticsABSTRACT
BACKGROUND: Orai1, the major store-operated Ca(2+) entry (SOCE) channel in platelets, is not only critical for enhancing diverse signaling pathways, but may also regulate receptor-operated Ca(2+) entry (ROCE). Dynamic coupling of the Orai1 signalosome to canonical transient receptor potential channels (TRPCs) has been suggested as an essential step in the activation of SOCE and ROCE. However, the functional significance of the biochemical interaction between Orai and TRPC isoforms remains controversial. OBJECTIVE: We aimed to elucidate the role of Orai1 in diacylglycerol (DAG)-mediated ROCE. METHODS: Trpc6(-/-) , Orai1(-/-) and Orai1(-/-) /Trpc6(-/-) mice were generated, and their platelets were analyzed. RESULTS: Thapsigargin (TG)-induced SOCE was further reduced in Orai1(-/-) /Trpc6(-/-) platelets as compared with Orai1(-/-) platelets, thus revealing that TG-induced signaling pathways can activate TRPC6. Thapsigargin-induced SOCE leads to enhanced phospholipase C and D activity in wild-type platelets. The activity of both enzymes was significantly reduced in Orai1(-/-) platelets upon TG stimulation, whereas receptor-induced phospholipase activity was not affected. Furthermore, TG-induced and glycoprotein VI-mediated thromboxane A2 release was strongly dependent on Orai1-mediated SOCE. CONCLUSION: The regulation of TRPC6 activity can occur independently of the physical interaction with Orai1. TRPC6 operates in crosstalk with Orai1 through Orai1-induced DAG production via phospholipase activation. Orai1-induced DAG production and thromboxane release amplify the second phase of Ca(2+) signaling in platelets.
Subject(s)
Calcium Channels/metabolism , Phospholipases/metabolism , TRPC Cation Channels/metabolism , Animals , Blood Platelets/cytology , Calcium/metabolism , Diglycerides/chemistry , Glycoproteins/chemistry , Liver/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , ORAI1 Protein , Platelet Aggregation , Signal Transduction , TRPC6 Cation Channel , Thapsigargin/metabolism , Thromboxane A2/metabolism , Time FactorsSubject(s)
Blood Platelets/metabolism , Hemostasis , Infarction, Middle Cerebral Artery/blood , Intracranial Hemorrhages/blood , Membrane Proteins/blood , Thrombosis/blood , Animals , Disease Models, Animal , Disease Progression , Hemostasis/genetics , Infarction, Middle Cerebral Artery/genetics , Infarction, Middle Cerebral Artery/pathology , Infarction, Middle Cerebral Artery/prevention & control , Intracranial Hemorrhages/genetics , Intracranial Hemorrhages/pathology , Membrane Proteins/deficiency , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Knockout , Thrombosis/genetics , Time FactorsABSTRACT
BACKGROUND: Platelet activation and aggregation are crucial for primary hemostasis, but can also result in occlusive thrombus formation. Agonist-induced platelet activation involves different signaling pathways leading to the activation of phospholipases, which produce second messengers. The role of phospholipase C (PLC) in platelet activation is well established, but less is known about the relevance of phospholipase D (PLD). OBJECTIVE AND METHODS: The aim of this study was to determine a potential function of PLD2 in platelet physiology. Thus, we investigated the function of PLD2 in platelet signaling and thrombus formation, by generating mice lacking PLD2 or both PLD1 and PLD2. Adhesion, activation and aggregation of PLD-deficient platelets were analyzed in vitro and in vivo. RESULTS: Whereas the absence of PLD2 resulted in reduced PLD activity in platelets, it had no detectable effect on the function of the cells in vitro and in vivo. However, the combined deficiency of both PLD isoforms resulted in defective α-granule release and protection in a model of FeCl3 -induced arteriolar thrombosis, effects that were not observed in mice lacking only one PLD isoform. CONCLUSION: These results reveal redundant roles of PLD1 and PLD2 in platelet α-granule secretion, and indicate that this may be relevant for pathologic thrombus formation.
