Diminished contact-dependent reinforcement of Syk activation underlies impaired thrombus growth in mice lacking Semaphorin 4D.
We recently reported that Semaphorin 4D (Sema4D) and its receptors are expressed on the platelet surface and showed that Sema4D((-/-)) mice have a selective defect in collagen-induced platelet aggregation and an impaired vascular injury response. Here we investigated the mechanisms involved, tested the role of platelet-platelet contacts in Sema4D-mediated events, and examined the relationship between Sema4D-dependent signaling and integrin α(IIb)β(3) outside-in signaling. The results show that spleen tyrosine kinase (Syk) activation, an early step in collagen signaling via the glycoprotein VI (GPVI)/FcRγ complex, is greatly reduced in Sema4D((-/-)) platelets and can be restored by adding soluble Sema4D. Earlier events, including FcRγ phosphorylation, occur normally; later events are impaired. In contrast, when engagement of α(IIb)β(3) was blocked, Sema4D((-/-)) and control platelets were indistinguishable in assays of Syk activation, adhesion, spreading on collagen, and activation of α(IIb)β(3). Finally, we found that, unlike the Sema4D knockout, α(IIb)β(3) blockade inhibited FcRγ phosphorylation and that stimulating aggregation with Mn(2+) failed to normalize Syk activation in the absence of Sema4D. Collectively, these results show that α(IIb)β(3) and Sema4D jointly promote collagen responses by amplifying Syk activation, partly by forming integrin-mediated contacts that enable the binding of Sema4D to its receptors and partly through integrin outside-in signaling. These 2 processes are interdependent, but distinguishable.
Wannemacher
Kenneth M
KM
Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA.
Zhu
Li
L
Jiang
Hong
H
Fong
Karen P
KP
Stalker
Timothy J
TJ
Lee
Dooyoung
D
Tran
Anh N
AN
Neeves
Keith B
KB
Maloney
Sean
S
Kumanogoh
Atsushi
A
Kikutani
Hitoshi
H
Hammer
Daniel A
DA
Diamond
Scott L
SL
Brass
Lawrence F
LF
D000818:Animals; D015703:Antigens, CD / Q000502:physiology*; D002118:Calcium / Q000378:metabolism; D003094:Collagen / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D016023:Integrins / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D051966:Phospholipase C gamma / Q000378:metabolism; D010766:Phosphorylation; D010974:Platelet Aggregation; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D039961:Semaphorins / Q000502:physiology*; D000072377:Syk Kinase; D013927:Thrombosis / Q000378:metabolism* / Q000473:pathology
D015703:Antigens, CD; C093224:CD100 antigen; D016023:Integrins; D047908:Intracellular Signaling Peptides and Proteins; D010980:Platelet Membrane Glycoproteins; D039961:Semaphorins; C098341:platelet membrane glycoprotein VI; D003094:Collagen; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; C000606897:Syk protein, mouse; D051966:Phospholipase C gamma; D002118:Calcium