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1.
J Thromb Haemost ; 12(8): 1364-71, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24909796

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/genetics
2.
J Thromb Haemost ; 12(4): 528-39, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24520961

ABSTRACT

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 Factors
3.
J Thromb Haemost ; 10(11): 2361-72, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22974101

ABSTRACT

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.


Subject(s)
Blood Platelets/metabolism , Phospholipase D/physiology , Adenosine Triphosphate/chemistry , Animals , Aorta/pathology , Bleeding Time , Blood Platelets/cytology , Cell Adhesion , Crosses, Genetic , Female , Hemostasis , Humans , Infarction, Middle Cerebral Artery/pathology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Platelet Activation , Platelet Factor 4/chemistry , Signal Transduction , Thrombosis/physiopathology , von Willebrand Factor/chemistry
4.
J Thromb Haemost ; 10(3): 419-29, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22176814

ABSTRACT

BACKGROUND: Platelet adhesion, activation and aggregation at sites of vascular injury are essential processes for primary hemostasis. Elevation of the intracellular Ca(2+) concentration is a central event in platelet activation but the underlying mechanisms are not fully understood. Store-operated calcium entry (SOCE) through Orai1 was shown to be the main Ca(2+) influx pathway in murine platelets, but there are additional non-store-operated Ca(2+) (non-SOC) and receptor operated Ca(2+) (ROC) channels expressed in the platelet plasma membrane. OBJECTIVE: Canonical transient receptor potential (TRPC) channel 6 is found both in human and murine platelets and has been proposed to mediate diacylglycerol (DAG) activated ROCE but also a role in the regulation of SOCE has been suggested. METHODS: To investigate the function of TRPC6 in platelet Ca(2+) signaling and activation, we analyzed platelets from mice deficient in TRPC6 using a wide range of in vitro and in vivo assays. RESULTS: In the mutant platelets, DAG activated Ca(2+) influx was found to be abolished. However, this did not significantly affect SOCE or agonist induced Ca(2+) responses. Platelet function in vitro and in vivo was also unaltered in the absence of TRPC6. CONCLUSION: Our results indicate that DAG activated ROCE is mediated exclusively by TRPC6 in murine platelets, but this Ca(2+) influx has no major functional relevance for hemostasis and thrombosis. Further, in contrast to previous suggestions, based on studies with human platelets, TRPC6 appears to play an insignificant role in the regulation of SOCE in murine platelets.


Subject(s)
Blood Platelets/metabolism , Calcium Signaling , Diglycerides/metabolism , Platelet Activation , TRPC Cation Channels/deficiency , Adenosine Diphosphate/metabolism , Animals , Blood Platelets/drug effects , C-Reactive Protein/metabolism , Calcium Channels/metabolism , Calcium Signaling/drug effects , Chlorides , Disease Models, Animal , Ferric Compounds , Gene Expression Regulation , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , ORAI1 Protein , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , RNA, Messenger/metabolism , Secretory Vesicles/drug effects , Secretory Vesicles/metabolism , Stromal Interaction Molecule 1 , TRPC Cation Channels/genetics , TRPC6 Cation Channel , Thrombin/metabolism , Thrombosis/blood , Thrombosis/chemically induced , Thrombosis/genetics , Time Factors
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