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Inhibition of αIIbβ3 Ligand Binding by an αIIb Peptide that Clasps the Hybrid Domain to the βI Domain of β3.

Lee WH, Schaffner-Reckinger E, Tsoukatos DC, Aylward K, Moussis V, Tsikaris V, Trypou P, Egot M, Baruch D, Kieffer N, Bachelot-Loza C - PLoS ONE (2015)

Bottom Line: The involvement of species-specific residues of the β3 hybrid domain (E356 and K384) and the β1 domain (E297) as well as an intrapeptide bond (pE315-pR317) were confirmed as important for this interaction by mutagenesis studies of αIIbβ3 expressed in CHO cells and native or substituted peptide inhibitory studies on platelet functions.Furthermore, NMR data corroborate the above results.Our findings provide insight into the important functional role of the αIIb β-ribbon in preventing integrin αIIbβ3 head piece opening, and highlight a potential new therapeutic approach to prevent integrin ligand binding.

View Article: PubMed Central - PubMed

Affiliation: SGC, University of Oxford, Oxford, United Kingdom.

ABSTRACT
Agonist-stimulated platelet activation triggers conformational changes of integrin αIIbβ3, allowing fibrinogen binding and platelet aggregation. We have previously shown that an octapeptide, p1YMESRADR8, corresponding to amino acids 313-320 of the β-ribbon extending from the β-propeller domain of αIIb, acts as a potent inhibitor of platelet aggregation. Here we have performed in silico modelling analysis of the interaction of this peptide with αIIbβ3 in its bent and closed (not swing-out) conformation and show that the peptide is able to act as a substitute for the β-ribbon by forming a clasp restraining the β3 hybrid and βI domains in a closed conformation. The involvement of species-specific residues of the β3 hybrid domain (E356 and K384) and the β1 domain (E297) as well as an intrapeptide bond (pE315-pR317) were confirmed as important for this interaction by mutagenesis studies of αIIbβ3 expressed in CHO cells and native or substituted peptide inhibitory studies on platelet functions. Furthermore, NMR data corroborate the above results. Our findings provide insight into the important functional role of the αIIb β-ribbon in preventing integrin αIIbβ3 head piece opening, and highlight a potential new therapeutic approach to prevent integrin ligand binding.

No MeSH data available.


Related in: MedlinePlus

CHO cell adhesion to fibrinogen in flow conditions.CHO cells expressing αIIbβ3 wild type (WT αIIbβ3), mutated on αIIb (αIIb3Mβ3) or on β3 (αIIbβ31M) were perfused over fibrinogen (A) at increasing shear rates (30 s-1, 50 s-1, 75 s-1 or 100 s-1) or (B) were preincubated 20 min with vehicle (0), 250 μM or 500 μM pYMESRADR peptide, or substituted control peptide (pYMESRAAR) and then perfused at a shear rate of 30 s-1. Adherent cells were counted after 12 min exposure to shear. (A) Results are expressed as the number of adherent cells/field, obtained by counting 10 fields. Bars represent mean values and the interquartile range of at least 3 experiments. (B) Adhesion of WT (left panel), αIIb3Mβ3 (middle panel) and αIIbβ31M (right panel) cells in the presence of peptides or vehicle was quantified (as described in methods section). The results are expressed as percentage of adhesion of each clone in presence of peptides relatively to corresponding cells in presence of vehicle (0) normalized to 100%. (n = 6; mean ± SEM). *P<0.05, **P<0.01
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pone.0134952.g006: CHO cell adhesion to fibrinogen in flow conditions.CHO cells expressing αIIbβ3 wild type (WT αIIbβ3), mutated on αIIb (αIIb3Mβ3) or on β3 (αIIbβ31M) were perfused over fibrinogen (A) at increasing shear rates (30 s-1, 50 s-1, 75 s-1 or 100 s-1) or (B) were preincubated 20 min with vehicle (0), 250 μM or 500 μM pYMESRADR peptide, or substituted control peptide (pYMESRAAR) and then perfused at a shear rate of 30 s-1. Adherent cells were counted after 12 min exposure to shear. (A) Results are expressed as the number of adherent cells/field, obtained by counting 10 fields. Bars represent mean values and the interquartile range of at least 3 experiments. (B) Adhesion of WT (left panel), αIIb3Mβ3 (middle panel) and αIIbβ31M (right panel) cells in the presence of peptides or vehicle was quantified (as described in methods section). The results are expressed as percentage of adhesion of each clone in presence of peptides relatively to corresponding cells in presence of vehicle (0) normalized to 100%. (n = 6; mean ± SEM). *P<0.05, **P<0.01

Mentions: It has been established that shear forces can increase the sensitivity of integrin αIIbβ3 adhesive interactions as demonstrated by the shear-dependent increase of adhesion of CHO cells expressing the PlA2 polymorphism of αIIbβ3 [30] and that αIIbβ3 activation is associated with a stabilization of integrin binding to fibrinogen under shear conditions between 30 s-1 and 100 s-1 [26]. In the present setting, we have demonstrated, as in previous work [26], that increasing shear rates results in decreased cell adhesion mediated by αIIbβ3. Interestingly, cells expressing the mutant integrins exhibited increased adhesion as compared to cells expressing wild type αIIbβ3. Notably, at 100 s-1, only cell clone αIIbβ31M showed residual adhesion. As the receptors displayed similar density and accessibility on the cell surface (S1 Fig), the differences in cell adhesion under shear relate most likely to a different activation state of the αIIbβ3 receptor expressed on the cell surface (Fig 6).


Inhibition of αIIbβ3 Ligand Binding by an αIIb Peptide that Clasps the Hybrid Domain to the βI Domain of β3.

Lee WH, Schaffner-Reckinger E, Tsoukatos DC, Aylward K, Moussis V, Tsikaris V, Trypou P, Egot M, Baruch D, Kieffer N, Bachelot-Loza C - PLoS ONE (2015)

CHO cell adhesion to fibrinogen in flow conditions.CHO cells expressing αIIbβ3 wild type (WT αIIbβ3), mutated on αIIb (αIIb3Mβ3) or on β3 (αIIbβ31M) were perfused over fibrinogen (A) at increasing shear rates (30 s-1, 50 s-1, 75 s-1 or 100 s-1) or (B) were preincubated 20 min with vehicle (0), 250 μM or 500 μM pYMESRADR peptide, or substituted control peptide (pYMESRAAR) and then perfused at a shear rate of 30 s-1. Adherent cells were counted after 12 min exposure to shear. (A) Results are expressed as the number of adherent cells/field, obtained by counting 10 fields. Bars represent mean values and the interquartile range of at least 3 experiments. (B) Adhesion of WT (left panel), αIIb3Mβ3 (middle panel) and αIIbβ31M (right panel) cells in the presence of peptides or vehicle was quantified (as described in methods section). The results are expressed as percentage of adhesion of each clone in presence of peptides relatively to corresponding cells in presence of vehicle (0) normalized to 100%. (n = 6; mean ± SEM). *P<0.05, **P<0.01
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pone.0134952.g006: CHO cell adhesion to fibrinogen in flow conditions.CHO cells expressing αIIbβ3 wild type (WT αIIbβ3), mutated on αIIb (αIIb3Mβ3) or on β3 (αIIbβ31M) were perfused over fibrinogen (A) at increasing shear rates (30 s-1, 50 s-1, 75 s-1 or 100 s-1) or (B) were preincubated 20 min with vehicle (0), 250 μM or 500 μM pYMESRADR peptide, or substituted control peptide (pYMESRAAR) and then perfused at a shear rate of 30 s-1. Adherent cells were counted after 12 min exposure to shear. (A) Results are expressed as the number of adherent cells/field, obtained by counting 10 fields. Bars represent mean values and the interquartile range of at least 3 experiments. (B) Adhesion of WT (left panel), αIIb3Mβ3 (middle panel) and αIIbβ31M (right panel) cells in the presence of peptides or vehicle was quantified (as described in methods section). The results are expressed as percentage of adhesion of each clone in presence of peptides relatively to corresponding cells in presence of vehicle (0) normalized to 100%. (n = 6; mean ± SEM). *P<0.05, **P<0.01
Mentions: It has been established that shear forces can increase the sensitivity of integrin αIIbβ3 adhesive interactions as demonstrated by the shear-dependent increase of adhesion of CHO cells expressing the PlA2 polymorphism of αIIbβ3 [30] and that αIIbβ3 activation is associated with a stabilization of integrin binding to fibrinogen under shear conditions between 30 s-1 and 100 s-1 [26]. In the present setting, we have demonstrated, as in previous work [26], that increasing shear rates results in decreased cell adhesion mediated by αIIbβ3. Interestingly, cells expressing the mutant integrins exhibited increased adhesion as compared to cells expressing wild type αIIbβ3. Notably, at 100 s-1, only cell clone αIIbβ31M showed residual adhesion. As the receptors displayed similar density and accessibility on the cell surface (S1 Fig), the differences in cell adhesion under shear relate most likely to a different activation state of the αIIbβ3 receptor expressed on the cell surface (Fig 6).

Bottom Line: The involvement of species-specific residues of the β3 hybrid domain (E356 and K384) and the β1 domain (E297) as well as an intrapeptide bond (pE315-pR317) were confirmed as important for this interaction by mutagenesis studies of αIIbβ3 expressed in CHO cells and native or substituted peptide inhibitory studies on platelet functions.Furthermore, NMR data corroborate the above results.Our findings provide insight into the important functional role of the αIIb β-ribbon in preventing integrin αIIbβ3 head piece opening, and highlight a potential new therapeutic approach to prevent integrin ligand binding.

View Article: PubMed Central - PubMed

Affiliation: SGC, University of Oxford, Oxford, United Kingdom.

ABSTRACT
Agonist-stimulated platelet activation triggers conformational changes of integrin αIIbβ3, allowing fibrinogen binding and platelet aggregation. We have previously shown that an octapeptide, p1YMESRADR8, corresponding to amino acids 313-320 of the β-ribbon extending from the β-propeller domain of αIIb, acts as a potent inhibitor of platelet aggregation. Here we have performed in silico modelling analysis of the interaction of this peptide with αIIbβ3 in its bent and closed (not swing-out) conformation and show that the peptide is able to act as a substitute for the β-ribbon by forming a clasp restraining the β3 hybrid and βI domains in a closed conformation. The involvement of species-specific residues of the β3 hybrid domain (E356 and K384) and the β1 domain (E297) as well as an intrapeptide bond (pE315-pR317) were confirmed as important for this interaction by mutagenesis studies of αIIbβ3 expressed in CHO cells and native or substituted peptide inhibitory studies on platelet functions. Furthermore, NMR data corroborate the above results. Our findings provide insight into the important functional role of the αIIb β-ribbon in preventing integrin αIIbβ3 head piece opening, and highlight a potential new therapeutic approach to prevent integrin ligand binding.

No MeSH data available.


Related in: MedlinePlus