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

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

Amino acid sequence alignments.

<p><b>(A)</b> Sequence alignments of the region surrounding amino acids 320, 321 and 353 from the human integrin αIIb subunit and αIIb from other species (upper sequences) or with other human α subunits (lower sequences); <b>(B)</b> Sequence alignments of the regions surrounding amino acids 297, 356 and 384 from the human integrin β3 subunit and β3 from other species (upper sequences) or with other human β subunits (lower sequences). Conserved residues between human αIIb or β3 and other sequences are shown with light shading.</p

Effect of octapeptides on platelet activation.

<p>Washed platelets (2.5x10⁸ platelets/ml) were preincubated with vehicle (NaCl 0.9%) or various peptides (A, B and D: 500 μM) for 5 min. Platelets were then treated with 0.1 U/ml thrombin (activated) or vehicle (resting) as described in “Experimental Procedures”. <b>(A)</b> LIBS (AP5 mAb) expression induced by vehicle, RGDS, ^pYMESRADR, RGDS + ^pYMESRADR, 317-substituted octapeptide (^pY³¹³MESAADR³²⁰), 319-substituted octapeptide (^pY³¹³MESRAAR³²⁰) or 320-substituted octapeptide (^pY³¹³MESRADA³²⁰) on resting or thrombin-activated platelets. AP5 binding was quantified by determining the fluorescence intensity (Geomean UA) of anti-mAb-phycoerythrin binding (n = 6; mean ± SEM). Isotype control binding (NI, first column) was performed in each experiment; <b>(B)</b> Flow cytometry analysis of anti-fibrinogen-FITC, PAC1-FITC, anti-CD62P-PE or isotype control (dashed black lines) antibody binding on resting (black lines) or activated platelets preincubated with vehicle (green lines), ^pYMESRADR octapeptide (red lines), or RGDS (blue lines); <b>(C)</b> Inhibitory effects of octapeptides (^pY³¹³MESRADR³²⁰: black; substituted ^pY³¹³MESRAAR³²⁰: grey or ^pM³¹⁴ESRADRK³²¹: dashed) on human washed platelet aggregation induced by thrombin (0.1 U/ml). Platelets were preincubated with vehicle (peptide 0 μM) or various concentrations of octapeptides and stimulated with thrombin for 5 min. Aggregation is expressed as a percentage of maximal light transmission measured at 5 min. Each point represents the mean (± SEM) of at least 4 experiments, *P<0.05, **P<0.01, ***P< 0,001 versus untreated platelets (peptide 0 μM); <b>(D)</b> Mean fluorescence intensity of PAC1-FITC binding on thrombin-activated platelets preincubated with vehicle, RGDS, ^pYMESRADR or with 317 substituted octapeptide (^pYMESAADR), 319 substituted octapeptide (^pYMESRAAR) or 320 substituted octapeptide (^pYMESRADA).</p

The β-ribbon YMESRADR sequence clasps αIIb to β3 in the bent-closed conformation.

<p>[LEFT] Two β3 subunit conformations have been observed in different crystal structures: bent-closed (green) (as seen in the structure of αIIbβ3—3FCS) and extended-open (purple) (as seen in the structure of αIIbβ3—1TY3/2VDK) while the αIIb β-propeller (yellow) position remains unchanged. The latch hairpin (blue, YMESRADR motif) is shown in the structure of the αIIb β-propeller; [RIGHT] Detail of the αIIb latch hairpin interaction with the bent-closed β3 (3FCS). The αIIb β-propeller domain (yellow) containing the β-ribbon and the YMESRADR motif (blue) engages the β3 hybrid domain (green). Two salt-bridges are established across the domains αIIb and β3: R320(αIIb)-E297(βI domain from β3) and K321(αIIb)-E356(hybrid domain from β3). An additional salt-bridge can be observed within the β-ribbon E315(αIIb)-R317(αIIb). In the same vicinity the αIIb β-propeller further engages with the β3 βI-domain through Y353(αIIb)-E297(β3). The interactions indicated above suggest that this region of the αIIb β-propeller plays a role in enforcing the positioning of the βI and hybrid domains when β3 is in its bent-closed conformation. The YMESRADR residues are shown as sticks with the carbon atoms coloured cyan, oxygen in red and nitrogen in blue. Residues from β3 involved in inter-subunit interactions are shown as sticks with white carbon atoms.</p

Free solution structures of octapeptides compared to the <i>in silico</i> model of ^pYMESRADR in its inhibitory conformation.

<p><b>(A)</b> Solution structure of native octapeptide ^pYMESRADR determined by NMR; <b>(B)</b><i>In silico</i> model of the octapeptide in its inhibitory conformation shown as sticks (as seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134952#pone.0134952.g003" target="_blank">Fig 3</a>) compared to the NMR ensemble of models of the ^pD319A substituted octapeptide shown as grey wires. Note that the intra-peptide salt-bridge between residues ^pE315- ^pR317 is present in both structures.</p