Tài liệu Báo cáo khoa học: Dual P2Y12 receptor signaling in thrombin-stimulated platelets –

Dual P2Y12 receptor signaling in thrombin-stimulated

platelets – involvement of phosphoinositide 3-kinase b

but not c isoform in Ca2+ mobilization and procoagulant

activity

Paola E. J. van der Meijden1

, Simone M. Schoenwaelder2

, Marion A. H. Feijge1

,

Judith M. E. M. Cosemans1

, Imke C. A. Munnix1

, Reinhard Wetzker3

, Regine Heller3

,

Shaun P. Jackson2 and Johan W. M. Heemskerk1

1 Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), University of Maastricht, the Netherlands

2 Australian Centre for Blood Diseases, Monash University, Alfred Medical Research Centre and Education Precinct (AMREP), Melbourne,

Australia

3 Institute of Molecular Cell Biology, University Hospital Jena, Germany

Platelets are activated at sites of vascular injury, and

then clump together to form a vaso-occlusive throm￾bus. Platelet activation is usually triggered by the

exposure of a thrombogenic surface such as collagen,

and continues by the availability of soluble agonists

that are derived from the injured vessel wall or the

activated platelets themselves. One of the most potent,

soluble platelet-activating agents is thrombin. Intravital

imaging studies of thrombus formation in damaged

mouse arteries indicate that thrombin is rapidly

formed at thrombotic sites via the tissue factor⁄fac￾tor VIIa pathway of coagulation [1]. This is confirmed

by inhibitory studies in various experimental models,

showing that thrombin generation plays a key, driving

Keywords

ADP; P2Y12; procoagulant activity; thrombin

Correspondence

J. W. M. Heemskerk, Department of

Biochemistry, University of Maastricht,

PO Box 616, 6200 MD Maastricht,

the Netherlands

Fax: +31 43 3884159

Tel: +31 43 3881671

E-mail: [email protected]

(Received 19 June 2007, revised 25 October

2007, accepted 26 November 2007)

doi:10.1111/j.1742-4658.2007.06207.x

During thrombus formation, thrombin, which is abundantly present at sites

of vascular injury, activates platelets in part via autocrine-produced ADP.

We investigated the signaling pathways by which thrombin and ADP in

synergy induced platelet Ca2+ elevation and procoagulant activity, and we

monitored the consequences for the coagulation process. Even at high

thrombin concentration, autocrine and added ADP enhanced and pro￾longed Ca2+ depletion from internal stores via stimulation of the P2Y12

receptors. This P2Y12-dependent effect was mediated via two distinct sig￾naling pathways. The first is enhanced Ca2+ mobilization by the inositol

1,4,5-trisphosphate receptors due to inhibition of protein kinase A. The

second pathway concerns prolonged activation of phosphoinositide

3-kinase (PI3-K) and phospholipase C. Experiments with phosphoinositide

3-kinase isoform-selective inhibitors and p110c deficient platelets demon￾strated that the phosphoinositide 3-kinase b and not the phosphoinositide

3-kinase c isoform is responsible for the prolonged Ca2+ response and for

the subsequent increases in procoagulant activity and coagulation. Taken

together, these results demonstrate a dual P2Y12-dependent signaling mech￾anism, which increases the platelet-activating effect of thrombin by prolon￾gation of Ca2+ elevation, thereby facilitating the coagulation process.

Abbreviations

AM, acetoxymethyl ester; FITC, fluorescein isothiocyanate; InsP3, inositol 1,4,5-trisphosphate; OG, Oregon green; PI3-K, phosphoinositide

3-kinase, SERCA, sarco- and endoplasmic reticulum Ca2+-ATPase; PKA, protein kinase A; PRP, platelet-rich plasma.

FEBS Journal 275 (2008) 371–385 ª 2007 The Authors Journal compilation ª 2007 FEBS 371