Sema6A exerts constitutive and PlxnA2-dependent cell-autonomous functions.

<p>(<b>A-F</b>) NIH3T3 cells expressing GFP alone (<b>A and B</b>), myc-Sema6A-FL (Sema6A; <b>B and E</b>), or myc-Sema6A-K393D (Sema6A-K393D; <b>C and F</b>) were treated with purified AP-Fc (AP; <b>A-C</b>) or PlxnA2-EC-Fc (PlxnA2-EC; <b>D-F</b>). Scale bar = 20 μm. (<b>G</b>) Graph represents the cell area in cells transfected with GFP, Sema6A or Sema6A-K393D and treated with AP or PlxnA2-EC; <i>n</i> = 100–200 cells per experimental condition. Data are expressed as mean ± s.e.m; ***<i>P</i> <0.001; Student’s <i>t</i>-test.</p

Sema6A cytoplasmic domain interacts with Abl and Mena.

<p>(<b>A</b>) Immunoprecipitations from untreated or PlxnA2-EC-treated COS-7 cells previously transfected with different combinations of myc-Sema6A, Abl-V5 and PlxnA2. Immunoprecipitations (Ip) were performed employing an anti-V5 antibody. Antibodies against V5, myc and PlxnA2 were used in the immunoblots (Ib). (<b>B</b>) Immunoprecipitations from untreated or PlxnA2-EC treated COS-7 cells previously transfected with different combinations of myc-Sema6A, Mena-V5 and PlxnA2. Immunoprecipitations (Ip) were performed employing an anti-myc antibody. Antibodies against V5, myc and PlxnA2 were used in the immunoblots (Ib). (<b>C</b>) and (<b>D</b>) Interactions were quantified by immunoblotting and densitometric analysis. Histograms represent quantification of interaction normalized to the amount of immunoprecipitated protein (Abelson-V5 for Sema6A-Abelson or Sema6A-myc for Sema6A-Mena) from three independent experiments. Students t-test *p<0,05 indicating signifiance. S6A-Abl/S6A-Abl+PlA2 <i>p</i> = 0.013, S6A-Abl-PlA2/S6A-Abl-PlA2+PlA2 <i>p</i> = 0.034. Errors bars represent standard error.</p

Sema6A reverse signaling is stimulated by multimerisation.

<p>(<b>A</b>) Scheme representing how the induced multi-aggregation of the cytosolic domain of Sema6A activates signaling pathways. A chimeric construct was engineered by putting in frame the entire cytosolic domain of Sema6A (Sema6A-cyt) together with 3 self-aggregation FKBP domains, and GFP. The chimeric peptide targets the plasma membrane by the myristoylating sequence located at the N-terminal. The application of the drug FK1012 triggers the chemically induced multimerisation (CIM) of Sema6A-cyt via the self-aggregation of FKBP. (<b>B</b>) Cerebellar neurons transfected with MF3-GFP or MF3-Sema6A-cyt-GFP (MF3-Sema6A-cyt), and treated with 500nM FK1012 (“CIM”). GFP-positive aggregates are indicated (arrowheads). Scale bar = 20μm. (<b>C</b>) Axonal shaft of cerebellar neurons transfected with myc-Sema6A-FL or MF3-Sema6A-cyt-GFP (MF3-Sema6A-cyt), and treated with PlxnA2-EC or CIM respectively. Scale bar = 5 μm. (<b>D</b>) and (<b>E</b>) Graphs summarising the myc-Sema6A-FL aggregation in myc-Sema6A-FL-expressing neurons treated with PlxnA2-EC at different time points. The aggregation is represented as the average distance between Sema6A clusters, or the average size of Sema6A clusters; <i>n</i> = 50–100 cells per time point. Data are expressed as mean ± s.e.m; ***<i>P</i> < 0.001; one-way ANOVA followed by Bonferroni multiple comparison test. (<b>F</b>) MF3-Sema6A-cyt-expressing granular neurons were cultured on NIH3T3 cell layers, and treated with CIM. Scale bar = 50 μm. (<b>G</b>) Graph representing the axonal length of MF3-Sema6A-cyt-expressing cerebellar neurons treated with CIM for 24 and 48h; <i>n</i> = 50–100 cells per experimental condition. Data are expressed as mean ± s.e.m; ***<i>P</i> < 0.001; one-way ANOVA followed by Bonferroni multiple comparison test.</p

Cell-contraction induced by PlxnA2-Sema6A interaction depends on Abl signaling.

<p>(<b>A-F</b>) NIH3T3 cells expressing GFP (<b>A</b> and <b>D</b>), Sema6A-FL (<b>B</b> and <b>E</b>), and Sema6A-∆Abl (<b>C</b> and <b>F</b>) were treated with purified AP-Fc (AP; <b>a-c</b>) or PlxnA2-EC-Fc (PlxnA2-EC; <b>D-F</b>). Scale bar = 20 μm. (<b>G</b>) Scheme illustrates that the cell contraction is via Abl signaling. (<b>H</b>) Graph represents the contraction of the cell area in NIH3T3 transfected with GFP, Sema6A or Sema6A-∆Abl and treated with AP or PlxnA2-EC; <i>n</i> = 100–200 cells per experimental condition. Data are expressed as mean ± s.e.m; ***<i>P</i> < 0.001; Student’s <i>t</i>-test.</p

Sema6A signaling reduces the axonal length of granular neurons.

<p>(<b>A-C</b>) GFP-expressing granular neurons were cultured on Control cells (<b>A</b>), Sema6A Cells (<b>B</b>), PlxnA2 Cells (<b>C</b>) and PlxnA2-A396E Cells (A396E Cells, <b>D</b>). Scale bar = 20 μm. (<b>E</b>) Graph represents the axonal length of granular neurons grown on different NIH3T3 layers; <i>n</i> = 100–200 cells per experimental condition. Data are expressed as mean ± s.e.m; ***<i>P</i> < 0.001; one-way ANOVA followed by Bonferroni multiple comparison test. (<b>F</b>) Graph represents the axonal length of <i>PlxnA2</i>^<i>+/+</i> (dark columns) and <i>PlxnA2</i>^<i>-/-</i> (light columns) granular neurons grown on NIH3T3 cells. Neurons were transfected with GFP alone or together with PlxnA2-FL, and cultured on control (Ctrl) or on Sema6A cell layers; <i>n</i> = 100–200 cells per experimental condition. Data are expressed as mean ± s.e.m; ***<i>P</i> < 0.001; Student’s <i>t</i>-test. (<b>G</b>) Graph represents the axonal length of <i>Sema6A</i>^<i>+/-</i> (dark columns) and <i>Sema6A</i>^<i>-/-</i> (light columns) granular neurons grown on NIH3T3 cells. Neurons were transfected with GFP alone or together with Sema6A-FL, and cultured on control (Ctrl) or on PlxnA2 cell layers; <i>n</i> = 100–200 cells per experimental condition. Data are expressed as mean ± s.e.m; ***<i>P</i> < 0.001; Student’s <i>t</i>-test. (<b>H-J</b>) Granular neurons transfected with GFP alone (<b>H</b> and <b>I</b>) or together with Sema6A-∆cyt (<b>J</b>), and grown on control or on PlxnA2 cells. Scale bar = 20 μm. (<b>K</b>) Graph represents the axonal length of <i>Sema6A</i>^<i>+/-</i> (dark columns) and <i>Sema6A</i>^<i>-/-</i> (light columns) granular neurons. Neurons were transfected with GFP alone or together with Sema6A-∆cyt, and cultured on control (Ctrl) or on PlxnA2 cell layers; <i>n</i> = 100–200 cells per experimental condition. Data are expressed as mean ± s.e.m; ***<i>P</i> < 0.001; Student’s <i>t</i>-test.</p

PlxnA2 interacts with Sema6A <i>in cis</i> and <i>in trans</i>.

<p>(<b>A</b>) Scheme summarising Sema6A-AP bindings. (<b>B-F</b>): COS-7 cells were transfected with the empty vector (mock; <b>B</b>), Sema6A-FL (<b>C</b>), PlxnA2-FL (<b>D</b>), PlxnA2-FL together with Sema6A-FL (<b>E</b>) or PlxnA2-A396E (<b>F</b>); and treated with Sema6A-AP. (<b>G</b>) Scheme summarising PlxnA2-AP bindings. (<b>H-K</b>): COS-7 cells were transfected with the empty vector (mock; <b>H</b>), Sema6A-FL (<b>I</b>), PlxnA2-FL (<b>J</b>) or PlxnA2-FL together with Sema6A-FL (<b>K</b>) and treated with PlxnA2-AP. (<b>L</b>) PlxnA2 immunoprecipitation (IP) from protein samples of COS-7 cells transfected with Sema6A-FL, PlxnA2-FL or Sema6A-FL together PlxnA2-FL. Antibodies against PlxnA2 and Sema6A were used in the immunoblots. (<b>M</b>) Sema6A IP from protein samples of COS-7 cells transfected with Sema6A-FL, PlxnA2-FL or Sema6A-FL together with PlxnA2-FL. (<b>N</b>) PlxnA2 IP from protein samples of mouse cerebellums or EGL explants. (<b>O</b>) PlxnA2 IP from protein samples of COS-7 cells transfected with PlxnA2-FL, Sema6A-∆cyt or Sema6A-∆cyt together with PlxnA2-FL. (<b>P</b>) PlxnA2 IP from protein samples of COS-7 cells transfected with PlxnA2-A396D, Sema6A-FL or Sema6A-FL together with PlxnA2-A396D. (<b>Q</b>) PlxnA2 IP from protein samples of COS-7 cells transfected with PlxnA2-FL, Sema6A-K393D or Sema6A-K393D together with PlxnA2-FL.</p

Pregnancy-specific glycoproteins bind integrin alpha IIb beta 3 and inhibit the platelet-fibrinogen interaction

Pregnancy-specific glycoproteins (PSGs) are immunoglobulin superfamily members encoded by multigene families in rodents and primates. In human pregnancy, PSGs are secreted by the syncytiotrophoblast, a fetal tissue, and reach a concentration of up to 400 mu g/ml in the maternal bloodstream at term. Human and mouse PSGs induce release of anti-inflammatory cytokines such as IL-10 and TGF beta 1 from monocytes, macrophages, and other cell types, suggesting an immunoregulatory function. RGD tri-peptide motifs in the majority of human PSGs suggest that they may function like snake venom disintegrins, which bind integrins and inhibit interactions with ligands. We noted that human PSG1 has a KGD, rather than an RGD motif. The presence of a KGD in barbourin, a platelet integrin alpha IIb beta 3 antagonist found in snake venom, suggested that PSG1 may be a selective alpha IIb beta 3 ligand. Here we show that human PSG1 binds alpha IIb beta 3 and inhibits the platelet - fibrinogen interaction. Unexpectedly, however, the KGD is not critical as multiple PSG1 domains independently bind and inhibit alpha IIb beta 3 function. Human PSG9 and mouse Psg23 are also inhibitory suggesting conservation of this function across primate and rodent PSG families. Our results suggest that in species with haemochorial placentation, in which maternal blood is in direct contact with fetal trophoblast, the high expression level of PSGs reflects a requirement to antagonise abundant (3 mg/ml) fibrinogen in the maternal circulation, which may be necessary to prevent platelet aggregation and thrombosis in the prothrombotic maternal environment of pregnancy

Multiple domains of human PSG1 bind the platelet integrin αIIbβ3.

<p><b>a,</b> Integrin αIIbβ3 (2µg purified protein; lanes 1–3) pulls down PSG1 in an <i>in vitro</i> binding assay (lane 1). Negative controls are Protein G agarose beads with (lane 2) and without (lane 4) αIIbβ3, and with rabbit IgG instead of PSG1 (lane 3). Similarly, αIIbβ3 from lysates of CHO cell line stably transfected with αIIbβ3 (lanes 7, 8), but not lysate of sham transfected CHO control cell line (lanes 5, 6) pulls down PSG1 in co-immunoprecipitation assays. Negative controls lack PSG1, but contain α-αIIbβ3 mAb bound to beads (lanes 5 & 7). Western blotted membranes were probed with α-αIIbβ3 mAb Sz22 (upper gel) and α-PSG1 mAb-5 (lower gel). <b>b,</b> Commercial purified integrin αIIbβ3 bound to Protein G agarose beads pulls down recombinant PSG1 (lane 1) and PSG1ΔN (lane 2). Negative controls lack PSG1 (lane 3) or αIIbβ3 (lane 4). Western blotted membranes were probed with α-αIIbβ3 mAb Sz22 (upper gel), and α-His-Tag pAb (lower gel) which detects tagged PSG1 and PSG1ΔN proteins. <b>c</b>, Representative image and pooled data of fluorescent PSG1 (PSG1–800) binding to CHO cell line stably transfected with αIIbβ3 compared to sham transfected CHO control cell line. Cell density was measured using SYTO60. Data are means of six independent experiments ± S.E.M. *, P<0.05, Paired Student’s t-test. <b>d,</b> Binding of the activation-dependent monoclonal antibody, PAC-1, to platelet αIIbβ3. Washed human platelets were preincubated with BSA or PSG1 at 200 µg/ml before the addition of PAC-1 antibody and the indicated platelet agonist: TRAP (4 µM), thromboxane mimetic U46619 (250 nM), ADP (10 µM) or epinephrine (25 µM). Data are means of four independent experiments ± S.E.M. *, P<0.05, Student’s t-test. <b>e,</b> Washed platelets adhere and spread extensively on fibrinogen-coated (20 µg/ml) glass slides but poorly on 1% BSA-coated slides. Pre-incubation of platelets with 200 µg/ml PSG1 significantly reduced platelet adhesion and spreading on fibrinogen. Permeabilized platelets were stained for polymerized F-actin with Alexa-488 fluorescein isothiocyanate phalloidin before visualisation using confocal microscopy. Representative images are shown. Scale bar is 20 µm. Graph shows quantification of platelet adhesion as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057491#s4" target="_blank">Methods</a>. Data are means of three independent experiments ± S.E.M. *, P<0.05, Student’s t-test.</p

PSG1 does not activate platelets.

<p><b>a,</b> Washed human platelets were treated at 37°C for 3 min with TRAP (4 µM) and/or PSG1 (200 µg/ml) for 2 min as indicated. Alternatively platelets remained untreated (resting). Platelet activation was assessed by analysis of the phosphotyrosine profile by western blotting with the antiphosphotyrosine mAb 4G10. Experiment was performed twice. <b>b, c</b> & <b>d,</b> In a similar series of experiments, three different markers of platelet degranulation were assessed. For ADP secretion assay (b), platelets were treated with 4 µM TRAP and/or PSG1 (100 µg/ml) for 3 min at 37^OC. For surface expression of CD62P (c) and CD63 (d) platelets were treated with 4 µM TRAP and/or PSG1 (100 µg/ml) for 10 min at RT as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057491#s4" target="_blank">Methods</a>. Alternatively platelets remained untreated (resting). Data represent the means of three independent experiments ± S.E.M.</p

Human and mouse PSGs inhibit the platelet – fibrinogen interaction.

<p>PSG-mediated inhibition of the platelet – fibrinogen interaction was measured by estimating binding of Oregon Green-conjugated fibrinogen (OgFg) to washed human platelets using FACS. Fibrinogen binding to TRAP-activated platelets is set at 100% and resting platelets at 0%. All assays were analysed over a four or five point dose range of PSG proteins and mutants, from ∼5–100 or 200 µg/ml, depending on protein molecular weight. For clarity, some results are reported as single dose molar concentration comparisons between proteins. Protein molecular weights were calculated from amino acid sequences with no adjustments for posttranslational modifications. <b>a,</b> Binding of OgFg to human platelets in the presence of human CEACAM1, human IgG, and increasing doses of recombinant wildtype human PSG1. 4 µM PSG1 is equivalent to 200 µg/ml protein. <b>b,</b> Binding of OgFg to human platelets in the presence of (left to right): wildtype PSG1 (KGD); PSG1 in which the KGD tri-peptide motif is replaced with RGE, or AAA; PSG1 with deletion of N-domain; PSG1 N-domain; PSG1 N-domain in which the KGD tri-peptide motif is replaced with AAA. All proteins were used at 2 µM concentration, equivalent to 100 µg/ml full-length PSG1 variants, 75 µg/ml for PSG1ΔN, and 38 µg/ml for PSG1N variants. <b>c</b> & <b>d,</b> Binding of OgFg to human platelets in the presence of increasing concentrations of recombinant human PSG9 and mouse Psg23, respectively. 2 µM PSG9 and 2 µM Psg23 is equivalent to 100 µg/ml and 110 µg/ml, respectively. <b>e,</b> Summary of domain structures and mutants of PSG proteins used (see Fig. S3 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057491#pone.0057491.s001" target="_blank">File S1</a> for sequences). <b>f,</b> Representative Coomassie-stained gels of protein used. For a - d, data are means of between three and seven independent experiments (detailed in main text) ± S.E.M. *, P<0.05; **, P<0.01; ***, P<0.001, nonparametric ANOVA with Dunnett’s multiple comparison post test.</p