Protease-activated receptor-induced Akt activation--regulation and possible function.
BACKGROUND
Thrombin induces the activation of the platelet serine/threonine kinase Akt. Akt activation is dependent on its phosphorylation at Thr308 and Ser473. The mechanism by which thrombin induces Akt phosphorylation is controversial, as is the role of Akt in platelet function.


OBJECTIVE
To investigate how protease-activated receptors (PARs) stimulate Akt and the role that Akt plays in human platelet function.


METHODS
Platelets were stimulated through PAR1 or PAR4. Specific inhibitors were used to evaluate, by Western blotting, signaling pathways regulating Akt phosphorylation, and the role of activated Akt was evaluated by aggregometry and flow cytometry.


RESULTS
Phospholipase C (PLC) controls Akt phosphorylation elicited by PARs. Stimulation of PAR1 or PAR4 resulted in rapid Akt phosphorylation, independently of secreted ADP and phosphatidylinositol-3-kinase (PI3K) activation. Akt phosphorylation approximately 60 s after PAR1 stimulation became entirely dependent on the purinergic receptor P2Y(12) and the activation of PI3K. In contrast, PAR4 partially sustained Akt phosphorylation independently of P2Y(12) and PI3K for up to 300 s. Pharmacologic inhibition of Akt reduced P-selectin expression and fibrinogen binding in platelets stimulated through PAR1, and delayed platelet aggregation in response to submaximal PAR1 or PAR4 stimulation, although aggregation at 300 s was unaffected.


CONCLUSIONS
Platelet PAR stimulation causes rapid Akt phosphorylation downstream of PLC, whereas with continuous stimulation, ADP and PI3K are required for maintaining Akt phosphorylation. Activated Akt regulates platelet function by modulating secretion and alpha(IIb)beta(3) activation.
Reséndiz
J C
JC
Wihuri Research Institute, Kalliolinnantie 4, Helsinki, Finland. julio.resendiz@wri.fi
Kroll
M H
MH
Lassila
R
R
D000244:Adenosine Diphosphate / Q000378:metabolism; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D015153:Blotting, Western; D004789:Enzyme Activation; D005340:Fibrinogen / Q000378:metabolism; D005434:Flow Cytometry; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D009842:Oligopeptides / Q000494:pharmacology; D019007:P-Selectin / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D010974:Platelet Aggregation / Q000187:drug effects; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020239:Receptor Cross-Talk; D044463:Receptor, PAR-1 / Q000819:agonists / Q000378:metabolism*; D018048:Receptors, Purinergic P2 / Q000378:metabolism; D058925:Receptors, Purinergic P2Y12; D018179:Receptors, Thrombin / Q000819:agonists / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013917:Thrombin / Q000378:metabolism*; D013997:Time Factors; D010738:Type C Phospholipases / Q000378:metabolism
D009842:Oligopeptides; D019007:P-Selectin; C548576:P2RY12 protein, human; C462999:PAR-1-activating peptide; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D047428:Protein Kinase Inhibitors; D044463:Receptor, PAR-1; D018048:Receptors, Purinergic P2; D058925:Receptors, Purinergic P2Y12; D018179:Receptors, Thrombin; C455901:alanyl-tyrosyl-prolyl-glycyl-lysyl-phenylalanine; D000244:Adenosine Diphosphate; D005340:Fibrinogen; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D010738:Type C Phospholipases; D013917:Thrombin; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; C112908:protease-activated receptor 4