Diminished contact-dependent reinforcement of Syk activation underlies impaired thrombus growth in mice lacking
Kenneth M Wannemacher1, Li Zhu, Hong Jiang
1Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA.
Abstract:
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.
Insights
Semaphorin 4D (Sema4D) and integrin α(IIb)β(3) work together to enhance collagen-induced platelet responses. This partnership amplifies spleen tyrosine kinase (Syk) activation, crucial for platelet aggregation and vascular injury repair.
Area of Science:
- Hematology
- Molecular Biology
- Cell Biology
Background:
- Semaphorin 4D (Sema4D) and its receptors are present on platelets.
- Sema4D-deficient mice exhibit impaired collagen-induced platelet aggregation and vascular injury response.
- The precise mechanisms linking Sema4D to platelet function remain unclear.
Purpose of the Study:
- To elucidate the mechanisms by which Sema4D influences collagen-mediated platelet activation.
- To investigate the role of platelet-platelet interactions in Sema4D signaling.
- To examine the interplay between Sema4D signaling and integrin α(IIb)β(3) outside-in signaling.
Main Methods:
- Analysis of spleen tyrosine kinase (Syk) activation in Sema4D-deficient platelets.
- Assessment of platelet adhesion, spreading, and aggregation under various conditions.
- Investigation of Fc receptor gamma (FcRγ) phosphorylation and integrin α(IIb)β(3) engagement.
- Use of soluble Sema4D and integrin α(IIb)β(3) blockade in experimental assays.
Main Results:
- Syk activation is significantly reduced in Sema4D-deficient platelets but can be restored by soluble Sema4D.
- FcRγ phosphorylation is normal in Sema4D-deficient platelets, but downstream events are impaired.
- Blocking integrin α(IIb)β(3) engagement rendered Sema4D-deficient and control platelets indistinguishable in several functional assays.
- Integrin α(IIb)β(3) blockade, unlike Sema4D deficiency, inhibited FcRγ phosphorylation, and Mn(2+)-induced aggregation failed to normalize Syk activation without Sema4D.
Conclusions:
- Integrin α(IIb)β(3) and Sema4D cooperate to promote collagen responses by amplifying Syk activation.
- This synergistic effect involves Sema4D binding to its receptors via integrin-mediated contacts and integrin outside-in signaling.
- These two pathways are interdependent yet distinct mechanisms contributing to platelet function.
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