Essential Role of Protein Kinase Cδ in Platelet Signaling, αIIbβ3 Activation, and Thromboxane A2 Release

Yacoub, Daniel; Théorêt, Jean François; Villeneuve, Louis; Abou-Saleh, Haissam; Mourad, Walid; Allen, Bruce G.; Merhi, Yahye

doi:10.1074/jbc.m604504200

Cited by 93 publications

(86 citation statements)

References 48 publications

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“…These data indicate that in platelets the stimulatory effects of PKC are regulated in a more distinct way than the suppressive effects of PKC, and they suggest that the latter are mediated by one or more nonclassical PKC isoforms. Mechanistically, these effects of Gö6976 are well explained by the recent observation that PKC␣ has a negative regulatory effect on Src kinases (45), the activity of which is of key importance in GPVI-induced signal transduction (23), although other authors propose that PKC␦ is required for collagen-induced phospholipase C activation (47). In HEK293 and COS1 cells, PKC␣ is recognized as a quickly responding element to local elevation in [Ca 2ϩ ] i (48).…”

Section: Discussionmentioning

confidence: 83%

Dual Role of Platelet Protein Kinase C in Thrombus Formation

Strehl

Munnix

Kuijpers

et al. 2007

Journal of Biological Chemistry

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Protein kinase C (PKC) isoforms regulate many platelet responses in a still incompletely understood manner. Here we investigated the roles of PKC in the platelet reactions implicated in thrombus formation as follows: secretion aggregate formation and coagulation-stimulating activity, using inhibitors with proven activity in plasma. In human and mouse platelets, PKC regulated aggregation by mediating secretion and contributing to ␣IIb␤3 activation. Strikingly, PKC suppressed Ca 2؉ signal generation and Ca 2؉ -dependent exposure of procoagulant phosphatidylserine. Furthermore, under coagulant conditions, PKC suppressed the thrombin-generating capacity of platelets. In flowing human and mouse blood, PKC contributed to platelet adhesion and controlled secretion-dependent thrombus formation, whereas it down-regulated Ca 2؉ signaling and procoagulant activity. In murine platelets lacking G q ␣, where secretion reactions were reduced in comparison with wild type mice, PKC still positively regulated platelet aggregation and down-regulated procoagulant activity. We conclude that platelet PKC isoforms have a dual controlling role in thrombus formation as follows: (i) by mediating secretion and integrin activation required for platelet aggregation under flow, and (ii) by suppressing Ca 2؉ -dependent phosphatidylserine exposure, and consequently thrombin generation and coagulation. This platelet signaling protein is the first one identified to balance the pro-aggregatory and procoagulant functions of thrombi.

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Section: Discussionmentioning

confidence: 83%

Dual Role of Platelet Protein Kinase C in Thrombus Formation

Strehl

Munnix

Kuijpers

et al. 2007

Journal of Biological Chemistry

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“…The serine/threonine kinases known to be activated in platelets in response to ADP and thrombin are PKCs, including PKC␦ (20,21), PKB/Akt, a downstream kinase of phosphatidylinositol 3-kinase (PI3K) (22)(23)(24), PKD, a PI3K and PKC-dependent kinase (25), and GSK3, which is also PI3K-dependent (26). Treatment of platelets with the PI3K inhibitors wortmannin or LY294002 did not affect levels of Rap1GAP2 serine 9 phosphorylation (Fig.…”

Section: Kinases Involved In Rap1gap2 Serine 9 Phosphorylation-mentioning

confidence: 95%

Cyclic Nucleotide-dependent Protein Kinases Inhibit Binding of 14-3-3 to the GTPase-activating Protein Rap1GAP2 in Platelets

Hoffmeister

Riha

Neumüller

et al. 2008

Journal of Biological Chemistry

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GTPase-activating proteins are required to terminate signaling by Rap1, a small guanine nucleotide-binding protein that controls integrin activity and cell adhesion. Recently, we identified Rap1GAP2, a GTPase-activating protein of Rap1 in platelets. Here we show that 14-3-3 proteins interact with phosphorylated serine 9 at the N terminus of Rap1GAP2. Platelet activation by ADP and thrombin enhances serine 9 phosphorylation and increases 14-3-3 binding to endogenous Rap1GAP2. Conversely, inhibition of platelets by endothelium-derived factors nitric oxide and prostacyclin disrupts 14-3-3 binding. These effects are mediated by cGMP-and cAMP-dependent protein kinases that phosphorylate Rap1GAP2 at serine 7, adjacent to the 14-3-3 binding site. 14-3-3 binding does not change the GTPase-activating function of Rap1GAP2 in vitro. However, 14-3-3 binding attenuates Rap1GAP2 mediated inhibition of cell adhesion. Our findings define a novel crossover point of activatory and inhibitory signaling pathways in platelets.Platelets are involved in many physiological and pathological events in the vascular system including hemostasis and thrombosis as well as inflammation, angiogenesis, and metastasis. Endothelium-derived messenger molecules nitric oxide (NO) and prostacyclin (PGI 2 ) 3 initiate two major inhibitory signaling pathways in platelets. NO and PGI 2 activate platelet guanylyl and adenylyl cyclases to produce cGMP and cAMP. In consequence cGMP and cAMP activate cGMP-and cAMP-dependent protein kinases (cGK/PKG and cAK/PKA) that phosphorylate substrate proteins leading to inhibition of platelet activation, adhesion and aggregation (1-3). To date only few substrates of cGK/cAKs in platelets have been characterized and the mechanisms mediating platelet inhibition downstream of the substrates are largely unknown. We and others (4, 5) have recently detected that cGK and cAK efficiently block agonistinduced formation of Rap1-GTP in platelets. Rap1 is a guanine nucleotide-binding protein that regulates integrin functions and plays a pivotal role in adhesion of many cell types (6 -9). In platelets Rap1 is required for adhesion, aggregation and thrombus formation (10). Because Rap1 controls integrin function and platelet aggregation, Rap1 inhibition might play a central role in platelet inhibition by NO and PGI 2 . Rap1 cycles between an active GTP-bound and an inactive GDP-bound conformation. The transition between these two states is controlled by unique guanine nucleotide exchange factors (GEF) and GTPase-activating proteins (GAP).In a previous study we identified Rap1GAP2, the only known GAP of Rap1 in platelets (11). Furthermore, in vitro experiments provided preliminary evidence for cGK-and cAK-mediated phosphorylation of Rap1GAP2 (11). Rap1GAP2 contains a central conserved GAP domain as well as large N-and C-terminal regions of unknown function. We hypothesized that these regions might be involved in protein/protein interactions and performed a genetic screening in yeast to identify Rap1GAP2-interacting proteins. In the p...

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“…TXA 2 is released as a positive feedback mediator when platelets are activated by physiological agonists such as thrombin, ADP, and 5-HT. 18,19) It has been shown that a TXA 2 receptor antagonist partially inhibits platelet aggregation induced by thrombin or ADP. 20,21) Therefore it is assumed that the 100% ethanol extract can inhibit platelet aggregation partially induced by other stimulants except a TXA 2 receptor agonist.…”

Section: Discussionmentioning

confidence: 99%