Investigation of the role of adjacent amino acids to the 313–320 sequence of the α_IIbsubunit on platelet activation and fibrinogen binding to α_IIbβ₃

Abstract

The platelet integrin receptor alphaIIbbeta3 plays a critical role in thrombosis and haemostasis by mediating interactions between platelets and several ligands, primarily fibrinogen. We have previously shown that the synthetic peptide YMESRADRKLAEVGRVYLFL corresponding to residues 313-332 of alphaIIb, is a potent inhibitor of platelet aggregation and fibrinogen binding to alphaIIbbeta3, interacting with fibrinogen rather than the receptor. Furthermore, we have demonstrated that the biological activities of the above peptide are due to the sequence YMESRADR, which corresponds to residues 313-320. By using new synthetic peptide analogues we investigated the structural characteristics responsible for the biological activity of YMESRADR as well the possible influence of the adjacent amino acids on the peptide's biological potency. According to our results, the synthetic octapeptide YMESRADR, is a potent inhibitor of platelet aggregation and P-selectin expression. Furthermore, YMESRADR inhibits fibrinogen binding but it does not significantly influence the binding of PAC-1 to ADP-activated platelets. The inhibitory potency of YMESRADR was gradually diminished by deleting the YMES sequence from the amino terminus and prolonging the carboxyl terminus of this peptide with the KLAE sequence. Extension of YMESRADR towards the amino terminus with the GAPL sequence (GAPLYMESRADR) does not modify the biological activity of YMESRADR. Furthermore, extension of GAPLYMESRADR at its carboxy terminus with the KLAE sequence (GAPLYMESRADRKLAE) significantly diminished its biological potency. Substitution of E315 with D significantly enhances antiaggregatory potency and completely abolishes the inhibitory effect on P-selectin expression. Importantly, the D315-containing peptides inhibit to a similar extent both fibrinogen and PAC-1 binding to activated alphaIIbbeta3 in contrast to the E315-containing peptide which only inhibits fibrinogen binding. In conclusion, the present study suggests that the YMESRADR sequence 313-320 of alphaIIb, is an important functional region of the insert connecting the beta2 and beta3 antiparallel beta-strands of the W5 blade of the alphaIIb subunit. Structural changes significantly modify the biological properties of this region.

Targeted molecular dynamics reveals overall common conformational changes upon hybrid domain swing‐out in β3 integrins

“…Specifically, these contacts are αIIb D319-β3 K384, αIIb R320-β3 E356, and αIIb K321-β3 E358. The first and third pairs are conserved in αVβ3 (αV D306-β3 K384 and αV K308-β3 E358), whereas in the latter the αIIb R320 residue is replaced by a glycine in αV (G307) 9,56-60. 2) β3 S300 (α5 helix) and β3 R360 (hybrid domain),32 whose Cβ distance increases from ∼9 to ∼34 Å; 3) Residues G349-R352 of the C-terminal region of α7 helix that contacts the hybrid domain;33-36 4) Residues S337-N339 and D126-S130 of the N-terminal portions of the α7 helix61,62 and the α1 helix,36,63-67 respectively, whose interactions are lost when the β6-α7 loop moves away from the ADMIDAS ion.…”

The human megakaryocytic cell line UT-7/TPO responds to platelet agonists with intracellular Ca2+elevation and P-selectin expression

“…In consequence, UT‐7/TPO is enriched in αIIb and β3, by up to 69% (Figure 4A). We further analysed the binding of anti‐activated αIIbβ3 antibody PAC1 (Mitsios et al, 2006) and the aggregation of UT‐7/TPO stimulated with thrombin. However, neither PAC‐1 binding (Figure 4B) nor aggregation (data not shown) in UT‐7/TPO was observed in spite of being stimulated with an excess concentration of thrombin (1 U/ml).…”

Exploration of the Antiplatelet Activity Profile of Betulinic Acid on Human Platelets

“…The surface expression of CD62P (P-selectin) and the PAC-1 binding to activated platelets was studied by flow cytometry in a FACSCalibur flow cytometer (Becton-Dickinson, San Jose, CA) using a slight modification of a technique previously described (28). Briefly, platelets were incubated in the presence or in the absence of 440 µM betulinic acid or 300 µM betulin with ADP, AA or TRAP (50 mM final concentration for each agonist) for 5 min at 37°C.…”