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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2007;27:422.)
© 2007 American Heart Association, Inc.

Thrombosis

GPVI Potentiation of Platelet Activation by Thrombin and Adhesion Molecules Independent of Src Kinases and Syk

Sascha C. Hughan; Craig E. Hughes; Owen J.T. McCarty; Edina Schweighoffer; Izoumroud Soultanova; Jerry Ware; Victor L.J. Tybulewicz; Steve P. Watson

From the Centre for Cardiovascular Sciences (S.C.H., C.E.H., O.J.T.M., S.P.W.), Institute of Biomedical Research, University of Birmingham, UK; the National Institute for Medical Research (E.S., V.L.J.T.), London, UK; Australian Centre for Blood Diseases (S.C.H.), Monash University, Melbourne, Victoria, Australia; and the Department of Physiology and Biophysics (I.S., J.W.), University of Arkansas for Medical Sciences, Little Rock. Current affiliation for O.J.T.M.: Department of Biomedical Engineering, Oregon Health and Science University, Portland, Ore.

Correspondence to Dr Sascha C. Hughan, Australian Centre for Blood Diseases, Monash University, Alfred Medical Research and Education Precinct, Melbourne, Victoria, Australia, 3004. E-mail Sascha.hughan{at}med.monash.edu.au

Objective— The present study investigates the role of Src and Syk tyrosine kinases in signaling by G-protein coupled and platelet adhesion receptors.

Methods and Results— Using Syk^–/– platelets or the Src kinase inhibitor PP2, we demonstrate a critical role for Src and Syk kinases in mediating lamellipodia formation on VWF, collagen, CRP, fibrinogen, and fibronectin. In all cases, the spreading defect was overcome by addition of thrombin. Conversely, platelet aggregation and _IIbß₃ activation induced by thrombin was similar to controls, arguing against a functional role for Src and Syk in _IIbß₃ activation. Unexpectedly, CRP potentiated integrin _IIbß₃ activation and platelet aggregation induced by subthreshold concentrations of thrombin in Syk^–/– platelets or in the presence of the Src kinase inhibitor PP2. Potentiation in the presence of PP2 was lost in the absence of FcR-chain or GPVI confirming that it was mediated through the immunoglobulin receptor. Further delineation of this PP2-resistant synergy revealed that PAR4 could trigger the enhanced response in combination with CRP.

Conclusions— We show that Syk is critical for lamellipodia formation on a range of immobilized proteins but that this can be overcome by addition of thrombin. Further, we reveal a novel role for GPVI in supporting thrombin-induced activation, independent of Syk and Src kinases.

We show a critical role for Syk in platelet spreading mediated by adhesion receptors that can be overcome by addition of G protein agonists. In addition, we demonstrate for the first time a synergistic pathway between GPVI and thrombin which promotes integrin _IIbß₃ activation independent of Syk and Src kinases.

Key Words: platelets • GPVI signaling • Src kinases • Syk • thrombin

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