Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Dec 16;116(25):5707-15.
doi: 10.1182/blood-2010-04-279943. Epub 2010 Sep 20.

Diminished contact-dependent reinforcement of Syk activation underlies impaired thrombus growth in mice lacking Semaphorin 4D

Kenneth M Wannemacher  1 , Li Zhu, Hong Jiang, Karen P Fong, Timothy J Stalker, Dooyoung Lee, Anh N Tran, Keith B Neeves, Sean Maloney, Atsushi Kumanogoh, Hitoshi Kikutani, Daniel A Hammer, Scott L Diamond, Lawrence F Brass
Affiliations

Diminished contact-dependent reinforcement of Syk activation underlies impaired thrombus growth in mice lacking Semaphorin 4D

Kenneth M Wannemacher et al. Blood. .

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.

PubMed Disclaimer

Figures

Figure 1
Figure 1
The Ca2+ response and phosphorylation of PLCγ2 after convulxin stimulation is decreased in Sema4D(−/−) platelets. (A) Platelets from matched Sema4D(+/+) and Sema4D(−/−) mice were loaded with Fura-2 and then stimulated with 0.5nM convulxin (CVX). (B) Summary data (mean ± SEM) from 6 studies with CVX. (C) Summary data from platelets stimulated with the PAR4 agonist peptide, AYPGQV (300μM, mean ± SEM, N = 6). N.S. = not significant. (D) Platelets from matched Sema4D(+/+) and Sema4D(−/−) mice were stimulated with 10nM CVX. Phosphorylation of PLCγ2 was measured at the times indicated. A representative immunoblot is shown and the results of 5 experiments are summarized (mean ± SEM). (E) Human platelets preincubated for 15 minutes with either a Sema4D blocking antibody (10 μg/mL) or an immunoglobulin control (10 μg/mL) were stimulated with 1 μg/mL collagen (mean ± SEM, N = 3). N.S. = not significant.
Figure 2
Figure 2
Decreased Syk phosphorylation in Sema4D(−/−) platelets. Platelets from matched Sema4D(+/+) and Sema4D(−/−) mice were stimulated with either (A) 5 μg/mL or (B) 10 μg/mL collagen. Lysates were prepared and immunoprecipitated with anti-Syk followed by immunoblotting with the anti-phosphotyrosine antibody, 4G10P (mean ± SEM, N = 3). N.S. = not significant.
Figure 3
Figure 3
FcRγ phosphorylation and the formation of the FcRγ/Syk complex proceed normally in the absence of Sema4D. Platelets from Sema4D(+/+) and Sema4D(−/−) mice were stimulated with collagen (5 μg/mL). (A) Lysates were immunoprecipitated with anti-FcRγ, followed by immunoblotting with the anti-phosphotyrosine antibody, 4G10P (mean ± SEM, N = 3). (B) Lysates were immunoprecipitated with anti-FcRγ followed by immunoblotting with anti-Syk (mean ± SEM, N = 6). N.S. = not significant.
Figure 4
Figure 4
Phosphorylation of Syk and FcRγ is diminished when platelet-platelet contacts are prevented. Platelets from Sema4D(−/−) and matched Sema4D(+/+) mice were incubated with Integrilin (10μM) plus aspirin (1mM) and apyrase (2 U/mL) for 30 minutes followed by collagen (2.5 μg/mL) for 2 minutes. (A) Immunoblot with anti-pSyk Y519/520 is shown. (B) FcRγ phosphorylation in Sema4D(+/+) platelets was detected by immunoprecipitating with anti-FcRγ followed by immunoblotting with anti-phosphotyrosine antibody, 4G10P. In both panels A and B, a representative experiment is shown and the results of 3 experiments are summarized (mean ± SEM). N.S. = not significant.
Figure 5
Figure 5
Promoting integrin engagement and Sema4D-mediated interactions increased Syk activation platelets. Washed platelets from Sema4D(+/+) and Sema4D(−/−) mice were supplemented with fibrinogen (150 μg/mL) and CaCl2 (1mM). Where indicated, MnCl2 (1mM) was added and the platelets were stirred for 7 minutes followed by a 2-minute incubation with or without collagen (2.5 μg/mL). (A) A representative immunoblot with anti-pSyk Y519/520 is shown, and the results of 4 experiments are summarized (mean ± SEM). (B) Representative aggregation traces for Mn2+-treated samples are shown. N.S. = not significant. (C) Washed platelets from Sema4D(+/+) and Sema4D(−/−) mice were incubated with 50 μg/mL rSema4D (recombinant Sema4D exodomain) for 10 minutes followed by collagen (2.5 μg/mL) for 2 minutes. A representative immunoblot with anti-pSyk Y519/520 is shown and results of 3 experiments are summarized (mean ± SEM). N.S. = not significant.
Figure 6
Figure 6
Fibrinogen binding, spreading on collagen and the formation of an initial platelet monolayer on a collagen-coated surface under flow proceeds normally in Sema4D(−/−) platelets. (A) Fibrinogen binding. Platelets from Sema4D(+/+) and Sema4D(−/−) mice were incubated with Alexa Fluor 488–labeled fibrinogen (100 μg/mL) and convulxin (CVX). Fibrinogen binding was measured by flow cytometry (mean ± SEM, N = 4-8). (B) Spreading of individual platelets on collagen. Platelets from Sema4D(+/+) and Sema4D(−/−) mice were deposited on glass coverslips coated with acid soluble type I collagen (0.1 mg/mL). Platelet spreading was quantified by reflection interference contrast microscopy (RICM). Representative images are shown and the results of 3 experiments are summarized (mean ± SEM). Scale bar equals 3 μm. (C-E) Platelets in PPACK-treated whole blood obtained from Sema4D(−/−) and matched Sema4D(+/+) mice were labeled with Alexa Fluor 488–conjugated anti-CD41 (αIIb) and perfused through a microfluidic flow chamber at 800 second−1. Platelet accumulation was detected in real time. Where indicated, a second, anti-CD41 antibody (Leo.H4, unlabeled) was used to block αIIbβ3 and prevent platelet aggregates from forming. (C-D) Video captures after 4 minutes of platelet accumulation. Fluorescence intensities for Sema4D(+/+) and Sema4D(−/−) platelets have been adjusted equally for presentation. (E) Changes in fluorescence intensity over time as platelets accumulated on the collagen-coated surface (mean ± SEM, N = 4). See supplemental videos.
Figure 7
Figure 7
Proposed contact-dependent role of Sema4D in GPVI signaling. (1) Based on work by others, clustering of GPVI leads to Src family kinase (SFK)–mediated phosphorylation of FcRγ resulting in the recruitment and (2) subsequent phosphorylation of Syk. Subsequent signaling through phospholipase Cγ leads to integrin activation and (3) the formation of stable, integrin-dependent contacts between platelets. (4) The data presented here suggest that this allows Sema4D to engage in trans with its receptors, amplifying Syk activation. It also allows outside-in signaling by the integrin to promote Syk phosphorylation, in part by increasing FcRγ phosphorylation (5).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Suzuki K, Kumanogoh A, Kikutani H. Semaphorins and their receptors in immune cell interactions. Nat Immunol. 2008;9(1):17–23. - PubMed
    1. Zhu L, Bergmeier W, Wu J, et al. Regulated surface expression and shedding support a dual role for semaphorin 4D in platelet responses to vascular injury. Proc Natl Acad Sci U S A. 2007;104(5):1621–1626. - PMC - PubMed
    1. Zhu L, Stalker TJ, Fong KP, et al. Disruption of SEMA4D ameliorates platelet hypersensitivity in dyslipidemia and confers protection against the development of atherosclerosis. Arterioscler Thromb Vasc Biol. 2009;29(7):1039–1045. - PMC - PubMed
    1. Tamagnone L, Artigiani S, Chen H, et al. Plexins are a large family of receptors for transmembrane, secreted, and GPI-anchored semaphorins in vertebrates. Cell. 1999;99(1):71–80. - PubMed
    1. Basile JR, Barac A, Zhu T, Guan KL, Gutkind JS. Class IV semaphorins promote angiogenesis by stimulating Rho-initiated pathways through plexin-B. Cancer Res. 2004;64(15):5212–5224. - PubMed

Publication types

MeSH terms