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Pregnancy-specific glycoproteins bind integrin αIIbβ3 and inhibit the platelet-fibrinogen interaction.

Shanley DK, Kiely PA, Golla K, Allen S, Martin K, O'Riordan RT, Ball M, Aplin JD, Singer BB, Caplice N, Moran N, Moore T - PLoS ONE (2013)

Bottom Line: The presence of a KGD in barbourin, a platelet integrin αIIbβ3 antagonist found in snake venom, suggested that PSG1 may be a selective αIIbβ3 ligand.Here we show that human PSG1 binds αIIbβ3 and inhibits the platelet - fibrinogen interaction.Unexpectedly, however, the KGD is not critical as multiple PSG1 domains independently bind and inhibit αIIbβ3 function.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University College Cork, Cork, Ireland.

ABSTRACT
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 ug/ml in the maternal bloodstream at term. Human and mouse PSGs induce release of anti-inflammatory cytokines such as IL-10 and TGFβ1 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 αIIbβ3 antagonist found in snake venom, suggested that PSG1 may be a selective αIIbβ3 ligand. Here we show that human PSG1 binds αIIbβ3 and inhibits the platelet - fibrinogen interaction. Unexpectedly, however, the KGD is not critical as multiple PSG1 domains independently bind and inhibit αIIbβ3 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.

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Human and mouse PSGs inhibit the platelet – fibrinogen interaction.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. a, 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, 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. c & d, 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. e, Summary of domain structures and mutants of PSG proteins used (see Fig. S3 in File S1 for sequences). f, 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.
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pone-0057491-g001: Human and mouse PSGs inhibit the platelet – fibrinogen interaction.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. a, 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, 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. c & d, 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. e, Summary of domain structures and mutants of PSG proteins used (see Fig. S3 in File S1 for sequences). f, 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.

Mentions: PSG1 is the only PSG with a KGD, rather than RGD, motif on the exposed F-G loop of the protein N-domain, a motif with selective activity towards platelet integrin αIIbβ3 in the barbourin disintegrin [25]. We tested whether it exhibits anti-thrombotic activity by determining whether recombinant PSG1 made in HEK293T cells inhibits binding of an Oregon Green conjugate of human fibrinogen to Thrombin Receptor-Activating Peptide (TRAP)-activated washed human platelets. Fibrinogen binding was inhibited in a dose-dependent manner with >90% inhibition at physiological doses of PSG1 (200 µg/ml; n = 7). No inhibition was observed using 200 µg/ml IgG purified from human blood, or using 200 µg/ml of the PSG1-related CEACAM1 protein produced in HEK293 cells (Fig. 1a). Similar results were obtained using platelets activated with 25 µM epinephrine, 250 nM thromboxane mimetic U46619, and 10 µM adenosine diphosphate (ADP) (Fig. S2 in File S1), consistent with PSG1 inhibiting a pathway common to all four activation reagents tested. To determine whether inhibition was mediated by the KGD motif, we replaced KGD with RGE, a motif found on the homologous F-G loop of several mouse Psg protein N1-domains and routinely used as a non-functional analogue of the integrin-binding RGD motif; and with AAA, which is expected to abolish putative KGD-mediated integrin-binding of the F-G loop (Fig. 1e). Both mutants showed similar inhibitory activity to wildtype PSG1 indicating that the KGD is not essential for PSG1-mediated inhibition of the platelet – fibrinogen interaction (Fig. 1b; n = 3 for each mutant).


Pregnancy-specific glycoproteins bind integrin αIIbβ3 and inhibit the platelet-fibrinogen interaction.

Shanley DK, Kiely PA, Golla K, Allen S, Martin K, O'Riordan RT, Ball M, Aplin JD, Singer BB, Caplice N, Moran N, Moore T - PLoS ONE (2013)

Human and mouse PSGs inhibit the platelet – fibrinogen interaction.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. a, 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, 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. c & d, 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. e, Summary of domain structures and mutants of PSG proteins used (see Fig. S3 in File S1 for sequences). f, 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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585349&req=5

pone-0057491-g001: Human and mouse PSGs inhibit the platelet – fibrinogen interaction.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. a, 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, 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. c & d, 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. e, Summary of domain structures and mutants of PSG proteins used (see Fig. S3 in File S1 for sequences). f, 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.
Mentions: PSG1 is the only PSG with a KGD, rather than RGD, motif on the exposed F-G loop of the protein N-domain, a motif with selective activity towards platelet integrin αIIbβ3 in the barbourin disintegrin [25]. We tested whether it exhibits anti-thrombotic activity by determining whether recombinant PSG1 made in HEK293T cells inhibits binding of an Oregon Green conjugate of human fibrinogen to Thrombin Receptor-Activating Peptide (TRAP)-activated washed human platelets. Fibrinogen binding was inhibited in a dose-dependent manner with >90% inhibition at physiological doses of PSG1 (200 µg/ml; n = 7). No inhibition was observed using 200 µg/ml IgG purified from human blood, or using 200 µg/ml of the PSG1-related CEACAM1 protein produced in HEK293 cells (Fig. 1a). Similar results were obtained using platelets activated with 25 µM epinephrine, 250 nM thromboxane mimetic U46619, and 10 µM adenosine diphosphate (ADP) (Fig. S2 in File S1), consistent with PSG1 inhibiting a pathway common to all four activation reagents tested. To determine whether inhibition was mediated by the KGD motif, we replaced KGD with RGE, a motif found on the homologous F-G loop of several mouse Psg protein N1-domains and routinely used as a non-functional analogue of the integrin-binding RGD motif; and with AAA, which is expected to abolish putative KGD-mediated integrin-binding of the F-G loop (Fig. 1e). Both mutants showed similar inhibitory activity to wildtype PSG1 indicating that the KGD is not essential for PSG1-mediated inhibition of the platelet – fibrinogen interaction (Fig. 1b; n = 3 for each mutant).

Bottom Line: The presence of a KGD in barbourin, a platelet integrin αIIbβ3 antagonist found in snake venom, suggested that PSG1 may be a selective αIIbβ3 ligand.Here we show that human PSG1 binds αIIbβ3 and inhibits the platelet - fibrinogen interaction.Unexpectedly, however, the KGD is not critical as multiple PSG1 domains independently bind and inhibit αIIbβ3 function.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University College Cork, Cork, Ireland.

ABSTRACT
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 ug/ml in the maternal bloodstream at term. Human and mouse PSGs induce release of anti-inflammatory cytokines such as IL-10 and TGFβ1 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 αIIbβ3 antagonist found in snake venom, suggested that PSG1 may be a selective αIIbβ3 ligand. Here we show that human PSG1 binds αIIbβ3 and inhibits the platelet - fibrinogen interaction. Unexpectedly, however, the KGD is not critical as multiple PSG1 domains independently bind and inhibit αIIbβ3 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.

Show MeSH
Related in: MedlinePlus