Non-redundant roles of phosphoinositide 3-kinase isoforms alpha and beta in glycoprotein VI-induced platelet signaling and thrombus formation.
Platelets are activated by adhesion to vascular collagen via the immunoglobulin receptor, glycoprotein VI (GPVI). This causes potent signaling toward activation of phospholipase Cgamma2, which bears similarity to the signaling pathway evoked by T- and B-cell receptors. Phosphoinositide 3-kinase (PI3K) plays an important role in collagen-induced platelet activation, because this activity modulates the autocrine effects of secreted ADP. Here, we identified the PI3K isoforms directly downstream of GPVI in human and mouse platelets and determined their role in GPVI-dependent thrombus formation. The targeting of platelet PI3Kalpha or -beta strongly and selectively suppressed GPVI-induced Ca(2+) mobilization and inositol 1,4,5-triphosphate production, thus demonstrating enhancement of phospholipase Cgamma2 by PI3Kalpha/beta. That PI3Kalpha and -beta have a non-redundant function in GPVI-induced platelet activation and thrombus formation was concluded from measurements of: (i) serine phosphorylation of Akt, (ii) dense granule secretion, (iii) intracellular Ca(2+) increases and surface expression of phosphatidylserine under flow, and (iv) thrombus formation, under conditions where PI3Kalpha/beta was blocked or p85alpha was deficient. In contrast, GPVI-induced platelet activation was insensitive to inhibition or deficiency of PI3Kdelta or -gamma. Furthermore, PI3Kalpha/beta, but not PI3Kgamma, contributed to GPVI-induced Rap1b activation and, surprisingly, also to Rap1b-independent platelet activation via GPVI. Together, these findings demonstrate that both PI3Kalpha and -beta isoforms are required for full GPVI-dependent platelet Ca(2+) signaling and thrombus formation, partly independently of Rap1b. This provides a new mechanistic explanation for the anti-thrombotic effect of PI3K inhibition and makes PI3Kalpha an interesting new target for anti-platelet therapy.
Gilio
Karen
K
Department of Biochemistry, Cardiovascular Research Institute Maastricht, University of Maastricht, 6200 MD Maastricht, The Netherlands.
Munnix
Imke C A
IC
Mangin
Pierre
P
Cosemans
Judith M E M
JM
Feijge
Marion A H
MA
van der Meijden
Paola E J
PE
Olieslagers
Servé
S
Chrzanowska-Wodnicka
Magdalena B
MB
Lillian
Rivka
R
Schoenwaelder
Simone
S
Koyasu
Shigeo
S
Sage
Stewart O
SO
Jackson
Shaun P
SP
Heemskerk
Johan W M
JW
D000818:Animals; D001792:Blood Platelets / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015539:Platelet Activation; D010974:Platelet Aggregation; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D020033:Protein Isoforms; D015398:Signal Transduction; D013927:Thrombosis / Q000378:metabolism / Q000473:pathology*
D010980:Platelet Membrane Glycoproteins; D020033:Protein Isoforms; C098341:platelet membrane glycoprotein VI; D019869:Phosphatidylinositol 3-Kinases; D002118:Calcium