Limits...
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.

Show MeSH

Related in: MedlinePlus

PSG1 does not activate platelets.a, 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, c & d, 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 Methods. Alternatively platelets remained untreated (resting). Data represent the means of three independent experiments ± S.E.M.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3585349&req=5

pone-0057491-g003: PSG1 does not activate platelets.a, 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, c & d, 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 Methods. Alternatively platelets remained untreated (resting). Data represent the means of three independent experiments ± S.E.M.

Mentions: We assessed whether PSG1 inhibits integrin function in platelets comparable to previously studied αIIbβ3 inhibitors [25]. We tested this by exposing washed platelets to 200 µg/ml PSG1 for 2 minutes at 37°C and measuring phosphotyrosine profiles on western blots of lysed platelets using anti-phosphotyrosine antibody (n = 2). PSG1 treatment did not increase protein phosphorylation compared to resting control treatment (Fig. 3a). However, pre-treatment of platelets with 200 µg/ml PSG1 for 2 minutes prior to TRAP activation reduced phosphorylation band intensity suggesting that PSG1 functionally inhibits platelet activation (Fig. 3a). This is consistent with our observations of PSG1-inhibited fibrinogen binding since phosphorylation events parallel integrin occupancy in platelets [29]. The integrin-specific nature of the PSG1-inhibition is also illustrated by the inability of PSG1 to affect other aspects of platelet activation. Activated platelets undergo degranulation causing release of ADP from dense granules, and increased surface expression of P-Selectin (CD62P) from alpha granules and CD63 from both lysosomes and dense granules [30]. We compared platelet ADP release and surface expression of CD62P and CD63 in TRAP activated platelets in the presence and absence of 100 µg/ml PSG1 (n = 3). Compared to robust responses to TRAP alone, PSG1 failed to affect any secretion responses demonstrating that the inhibitory effects of PSG1 on platelets are integrin αIIbβ3-specific (Fig. 3b, c, d).


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)

PSG1 does not activate platelets.a, 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, c & d, 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 Methods. Alternatively platelets remained untreated (resting). Data represent the means of three independent experiments ± S.E.M.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0057491-g003: PSG1 does not activate platelets.a, 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, c & d, 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 Methods. Alternatively platelets remained untreated (resting). Data represent the means of three independent experiments ± S.E.M.
Mentions: We assessed whether PSG1 inhibits integrin function in platelets comparable to previously studied αIIbβ3 inhibitors [25]. We tested this by exposing washed platelets to 200 µg/ml PSG1 for 2 minutes at 37°C and measuring phosphotyrosine profiles on western blots of lysed platelets using anti-phosphotyrosine antibody (n = 2). PSG1 treatment did not increase protein phosphorylation compared to resting control treatment (Fig. 3a). However, pre-treatment of platelets with 200 µg/ml PSG1 for 2 minutes prior to TRAP activation reduced phosphorylation band intensity suggesting that PSG1 functionally inhibits platelet activation (Fig. 3a). This is consistent with our observations of PSG1-inhibited fibrinogen binding since phosphorylation events parallel integrin occupancy in platelets [29]. The integrin-specific nature of the PSG1-inhibition is also illustrated by the inability of PSG1 to affect other aspects of platelet activation. Activated platelets undergo degranulation causing release of ADP from dense granules, and increased surface expression of P-Selectin (CD62P) from alpha granules and CD63 from both lysosomes and dense granules [30]. We compared platelet ADP release and surface expression of CD62P and CD63 in TRAP activated platelets in the presence and absence of 100 µg/ml PSG1 (n = 3). Compared to robust responses to TRAP alone, PSG1 failed to affect any secretion responses demonstrating that the inhibitory effects of PSG1 on platelets are integrin αIIbβ3-specific (Fig. 3b, c, d).

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