Congress of the International Society on Thrombosis and Haemostasis

Akt regulates weak-agonist mediated Rap1b activation via a GSK-3 independent signalling pathway in platelets

Abstract number: PP-TH-014

Sumathipala¹ R.N., Patel² K.V., Rahman¹ S., Nishino¹ Y., Webb¹ I.G., Rangarajan² S., Kaiser³ W.J., Gibbins³ J.M., Patel⁴ Y.M.

¹¹Cardiology, Cardiovascular Division, Kings College London ²²Centre for Haemostasis and Thrombosis, St Thomas’ Hospital, London ³³School of Biological Sciences, University of Reading, Reading ⁴⁴Cardiovascular Division, Kings College London, London, UK

How-to-cite Sumathipala RN, Patel KV, Rahman S, Nishino Y, Webb IG, Rangarajan S, Kaiser WJ, Gibbins JM, Patel YM. Akt regulates weak-agonist mediated Rap1b activation via a GSK-3 independent signalling pathway in platelets. Journal of Thrombosis and Haemostasis 2009; Volume 7, Supplement 2: Abstract PP-TH-014

The role GSK-3 plays in regulating weak agonist-induced Rap1B activation was investigated using two approaches. Firstly, Rap1B activation and GSK-3 inactivation were examined in the platelets of a patient in whom we have previously demonstrated defective post-receptor Gi-signalling (Patel et al 2003; Blood 101:4828–4835). Secondly, Rap1B activation was examined in GSK-3 Knockin (KI) mouse platelets. The targeting strategy resulted in dual GSK-3alpha/beta KI mice, in which the Akt phosphorylation sites on GSK-3alpha(Ser21) and GSK-3beta(Ser9) were changed to Ala thus rendering both isoforms incapable of being inactivated by Akt. In the patient's platelets, Rap1B activation was suppressed in response to stimulation with either a combination of ADP (10 μM) plus adrenaline (10 μM) or low dose thrombin (0.1 U/mL) respectively. In normal platelets, preincubation with the P2Y12 receptor antagonist AR-C67085MX (10 μM;AR-C) also resulted in the inhibition of the response indicating a role for Gi signalling mediated by released ADP. Thrombin induced Akt activation and GSK-3 inactivation, as assessed by Akt (Ser473) and GSK3beta (Ser9) phosphorylation, were almost totally suppressed in the patient's platelets. AR-C pre-treatment of normal platelets resulted in the inhibition of agonist-induced responses, confirming a role for Gi signalling induced by released ADP in regulating both events. Weak agonist-mediated Rap1B activation was also investigated in GSK-3 KI mouse platelets. Platelets from WT or KI mice were stimulated with either ADP plus adrenaline, AYPGKF (1 mM) or high dose thrombin (1 U/mL) respectively and responses compared. The level of Rap1B activation by these agonists in GSK-3 KI mice platelets was comparable with responses in WT platelets. Pre-treatment of either WT or GSK-3 KI mice platelets with the GSK-3 inhibitor SB415286 (50 μM) also had no effect on ADP plus adrenaline-mediated Rap1B activation. In comparison, pre–treatment with the Akt inhibitor SH-6 (20 μM) resulted in the inhibition (> 70%) of the response in both WT and GSK-3 KI platelets indicating an important role for Akt in regulating this event. This study indicates that Akt but not GSK-3 plays a role in regulating weak agonist induced Rap1B activation via a novel signalling pathway.

Disclosure of interest: none declared.

To cite this abstract use the following format:

Journal of Thrombosis and Haemostasis 2007; Volume 5, Supplement 2: abstract number

Session Details

Date:	Unpresented
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Session name:	ISTH2009
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