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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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Multiple domains of human PSG1 bind the platelet integrin αIIbβ3.a, Integrin αIIbβ3 (2µg purified protein; lanes 1–3) pulls down PSG1 in an in vitro 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, 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. c, 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. d, 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. e, 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 Methods. Data are means of three independent experiments ± S.E.M. *, P<0.05, Student’s t-test.
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pone-0057491-g002: Multiple domains of human PSG1 bind the platelet integrin αIIbβ3.a, Integrin αIIbβ3 (2µg purified protein; lanes 1–3) pulls down PSG1 in an in vitro 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, 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. c, 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. d, 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. e, 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 Methods. Data are means of three independent experiments ± S.E.M. *, P<0.05, Student’s t-test.

Mentions: The dispensability of the KGD motif led us to question whether anti-thrombotic activity might be mediated by a mechanism other than antagonism of αIIbβ3 integrin, for example, by direct binding of PSG1 to fibrinogen. To rule this out, we tactically changed from fibrinogen competition assays and focused on the specificity of the PSG1– integrin interaction using Co-IP. We showed that PSG1 was pulled down by αIIbβ3 integrin from a purified preparation and from a CHO cell line stably transfected with human αIIbβ3 (Fig. 2a). No signal was obtained following pull-down using an empty vector-transfected control CHO cell line. In a further co-IP experiment, both wildtype PSG1 and PSG1ΔN were pulled down by purified αIIbβ3 supporting the existence of anti-αIIbβ3 activity in one or more of the A1, A2 or B2 domains (Fig. 2b). In similar experiments, we showed that recombinant full-length PSG1 and the PSG1 N-domain protein, with and without the KGD → AAA mutation, were pulled down by purified αIIbβ3 integrin, indicating that the KGD motif is dispensable for integrin binding by the PSG1 N-domain (Fig. S5 in File S1).


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)

Multiple domains of human PSG1 bind the platelet integrin αIIbβ3.a, Integrin αIIbβ3 (2µg purified protein; lanes 1–3) pulls down PSG1 in an in vitro 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, 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. c, 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. d, 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. e, 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 Methods. Data are means of three independent experiments ± S.E.M. *, P<0.05, Student’s t-test.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585349&req=5

pone-0057491-g002: Multiple domains of human PSG1 bind the platelet integrin αIIbβ3.a, Integrin αIIbβ3 (2µg purified protein; lanes 1–3) pulls down PSG1 in an in vitro 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, 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. c, 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. d, 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. e, 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 Methods. Data are means of three independent experiments ± S.E.M. *, P<0.05, Student’s t-test.
Mentions: The dispensability of the KGD motif led us to question whether anti-thrombotic activity might be mediated by a mechanism other than antagonism of αIIbβ3 integrin, for example, by direct binding of PSG1 to fibrinogen. To rule this out, we tactically changed from fibrinogen competition assays and focused on the specificity of the PSG1– integrin interaction using Co-IP. We showed that PSG1 was pulled down by αIIbβ3 integrin from a purified preparation and from a CHO cell line stably transfected with human αIIbβ3 (Fig. 2a). No signal was obtained following pull-down using an empty vector-transfected control CHO cell line. In a further co-IP experiment, both wildtype PSG1 and PSG1ΔN were pulled down by purified αIIbβ3 supporting the existence of anti-αIIbβ3 activity in one or more of the A1, A2 or B2 domains (Fig. 2b). In similar experiments, we showed that recombinant full-length PSG1 and the PSG1 N-domain protein, with and without the KGD → AAA mutation, were pulled down by purified αIIbβ3 integrin, indicating that the KGD motif is dispensable for integrin binding by the PSG1 N-domain (Fig. S5 in File S1).

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