Phosphorylation and Dephosphorylation Mechanisms in Platelet Function: a Tightly Regulated Balance

Lévy-Toledano, S; Gallet, Carole; Nadal, Florence; Bryckaert, Marijke; Maclouf, Jacques; Rosa, Jean‐Philippe

doi:10.1055/s-0038-1657531

Cited by 40 publications

(30 citation statements)

References 55 publications

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“…However, platelet interactions with a collagen-rich surface are related to deep vascular damage and acute thrombotic events, which could trigger a more intense cascade of signalling mechanisms [23]. Tyrosine phosphorylation of proteins is one of the signalling mechanisms that regulates cell responses [25], and which has been extensively studied in platelets activated in suspension [5,26]. Our present study has considered that platelets fulfil their main function by interacting with the subendothelium under flow conditions.…”

Section: Discussionmentioning

confidence: 99%

Primary arrest of circulating platelets on collagen involves phosphorylation of Syk, cortactin and focal adhesion kinase: studies under flow conditions

Arderiu

Díaz‐Ricart

Buckley

et al. 2002

Biochemical Journal

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After a vessel wall injury, platelets adhere to the subendothelium following a sequence of events: arrest of single platelets on the surface, progression to platelet spreading and final aggregation. Primary arrest of circulating platelets on subendothelial components occurs through platelet glycoprotein (GP) Ib and collagen receptors; then platelets spread and aggregate through a GPIIb-IIIa-dependent mechanism. A series of strategies were applied to analyse the tyrosine-phosphorylation mechanisms occurring at the different stages of platelet adhesion on subendothelial components under flow conditions, with special attention to primary arrest. To evaluate spread platelets, samples were exposed to acetylsalicylic acid, which blocks aggregate formation. To study single platelets in contact, a monoclonal antibody specific for GPIIb-IIIa was used to prevent platelet spreading and further aggregation. This experimental situation was also investigated using blood from two patients with Glanzmann's thrombasthenia (i.e. lacking GPIIb-IIIa). Results demonstrated that blockade of both spreading and aggregation results in significant changes in the tyrosine-phosphorylation patterns. Arrest of single platelets on collagen-rich surfaces resulted in phosphorylation of p125, identified as focal adhesion kinase (FAK), the 80/85kDa doublet (cortactin), and p72, identified as Syk. Arrest of single platelets on von Willebrand factor as adhesive substrate showed that interaction through GPIb induces Syk phosphorylation, but not that of cortactin and FAK. Our data indicate that the initial arrest of platelets on subendothelial components involves Syk phosphorylation, which seems to be GPIb-dependent, and this is followed by activation and phosphorylation of cortactin and FAK. These processes seem to occur before GPIIb-IIIa becomes activated.

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Section: Discussionmentioning

confidence: 99%

Primary arrest of circulating platelets on collagen involves phosphorylation of Syk, cortactin and focal adhesion kinase: studies under flow conditions

Arderiu

Díaz‐Ricart

Buckley

et al. 2002

Biochemical Journal

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“…In platelets, p38 MAPK is activated by stress, such as heat and osmotic shock, arsenite, H 2 O 2 , ␣-thrombin, collagen, and thromboxane analog (11,12), and is involved in the phosphorylation of [Ca 2ϩ ]-dependent cytosolic phospholipase A 2 (cPLA 2 ), with subsequent production of TXB 2 ( Figure 1). Thrombin has also been shown to induce phosphorylation of ERK-1/2, involving protein kinase C (PKC), phospholipase C␤ (PLC␤), and the intracellular mobilization of [Ca 2ϩ ] (Figure 1) (13)(14)(15). Although several studies have shown that aPL enhance platelet activation in vitro in the presence of low doses of agonists (ADP, thrombin, collagen, or TRAP) (7)(8)(9)(10)16), the intracellular events involved in this process are not understood.…”

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confidence: 99%

Intracellular events in platelet activation induced by antiphospholipid antibodies in the presence of low doses of thrombin

Vega-Ostertag

Harris

Pierangeli

2004

Arthritis & Rheumatism

118

102

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Objective. Thrombosis and thrombocytopenia are features of the antiphospholipid syndrome (APS), suggesting that antiphospholipid antibodies (aPL) may bind platelets, causing activation and aggregation of platelets and thrombosis. The intracellular events involved in aPL-mediated platelet activation are not fully understood and are therefore the subject of this study.Methods. IgG fractions and their F(ab) 2 fragments were purified from the sera of 7 patients with APS and from the pooled sera of 10 healthy subjects (as controls). Phosphorylation of p38 MAPK, ERK-1/2, and [Ca 2؉ ]-dependent cytosolic phospholipase A 2 (cPLA 2 ) was determined in lysates of washed platelets pretreated with low doses of thrombin and aPL or control IgG or their F(ab) 2 fragments, by immunoblot. The effects of aPL on platelet aggregation in the presence or absence of a p38 MAPK inhibitor, SB203580, were examined. Thromboxane B 2 (TXB 2 ) production was detected by enzyme-linked immunosorbent assay on gel-filtered platelets treated with aPL and thrombin, with or without SB203580. Calcium mobilization studies were done utilizing a fluorometric assay.Results. Treatment of platelets with IgG aPL, or their F(ab) 2 fragments, in conjunction with subactivating doses of thrombin resulted in a significant increase in phosphorylation of p38 MAPK. Neither the IgG aPL nor their F(ab) 2 fragments increased significantly the phosphorylation of ERK-1/2 MAPKs. Furthermore, pretreatment of platelets with SB203580 completely abrogated the aPL-mediated enhanced aggregation of the platelets. Platelets treated with F(ab) 2 aPL and thrombin produced significantly larger amounts of TXB 2 when compared with controls, and this effect was completely abrogated by treatment with SB203580. In addition, cPLA 2 was also significantly phosphorylated in platelets treated with thrombin and F(ab) 2 aPL. There were no significant changes in intracellular [Ca 2؉ ] when platelets were treated with aPL and low doses of thrombin.Conclusion. The data strongly indicate that aPL in the presence of subactivating doses of thrombin induce the production of TXB 2 mainly through the activation of p38 MAPK and subsequent phosphorylation of cPLA 2 . The ERK-1/2 pathway does not seem to be involved in this process, at least in the early stages of aPL-mediated platelet activation.

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“…5). In these experimental settings, signaling as mediated by ␣ IIb ␤ 3 was shown to involve tyrosine kinases, including Syk, Fak, and Src, as well as tyrosine phosphatases, such as PTP1B (5,12,13). In contrast, signaling associated with clot retraction by platelets has been the subject of only a few reports.…”

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confidence: 99%

Fibrin Clot Retraction by Human Platelets Correlates with αIIbβ3Integrin-dependent Protein Tyrosine Dephosphorylation

Osdoit

Rosa

2001

Journal of Biological Chemistry

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We have analyzed tyrosine phosphorylation associated with retraction of the fibrin clot by washed platelets in purified fibrinogen. Retraction was dependent on integrin ␣ IIb ␤ 3 , based on absence of retraction of ␣ IIb ␤ 3 -deficient thrombasthenic platelets. However, only a subset of ␣ IIb ␤ 3 -blocking antibodies or peptides were able to inhibit retraction, suggesting a differential engagement of ␣ IIb ␤ 3 in fibrin clot retraction versus aggregation. Immunoblotting demonstrated a phosphorylated protein pattern comparable with aggregation at early time points. However, as opposed to aggregation, tyrosine phosphorylation decreased rapidly in parallel to retraction (up to 60 min). Dephosphorylation was ␣ IIb ␤ 3 -dependent, since it was blocked by ␣ IIb ␤ 3 -specific inhibitors and was absent in thrombasthenic platelets. Inhibition of platelet clot retraction by phenyl-arsine oxide and peroxovanadate, suggested a role for tyrosine phosphatases. Cytochalasin D and E (5 M) blocked fibrin clot retraction and tyrosine dephosphorylation, suggesting regulation by actin cytoskeleton assembly. Tyrosine phosphatase activities were found associated with clot retraction using the "in-gel" tyrosine phosphatase assay; however, none were ␣ IIb ␤ 3 -dependent. An 85-kDa protein and to a lesser degree "Src" showed the closest dose-dependent correlation between inhibition of tyrosine dephosphorylation and inhibition of retraction. We thus postulate that ␣ IIb ␤ 3 engagement in fibrin clot retraction drives, in an actin cytoskeleton-dependent manner, the interaction of tyrosine phosphatases and of the tyrosine-phosphorylated substrates 85-kDa protein and Src, the dephosphorylation of which regulates the force generation and/or transmission required for full contraction of the fibrin matrix.

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Phosphorylation and Dephosphorylation Mechanisms in Platelet Function: a Tightly Regulated Balance

Cited by 40 publications

References 55 publications

Primary arrest of circulating platelets on collagen involves phosphorylation of Syk, cortactin and focal adhesion kinase: studies under flow conditions

Primary arrest of circulating platelets on collagen involves phosphorylation of Syk, cortactin and focal adhesion kinase: studies under flow conditions

Intracellular events in platelet activation induced by antiphospholipid antibodies in the presence of low doses of thrombin

Fibrin Clot Retraction by Human Platelets Correlates with αIIbβ3Integrin-dependent Protein Tyrosine Dephosphorylation

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