Inhibition of platelet activation by peptide analogs of theβ₃-intracellular domain of platelet integrinα_IIbβ₃conjugated to the cell-penetrating peptide Tat(48–60)

Abstract

Activation of the platelet integrin-receptor alpha(IIb)beta(3) is the final pathway of platelet aggregation, regardless of the initiating stimulus. Many studies suggest that there are several cytoplasmic proteins such as talin and beta(3)-endonexin that bind to N(744)PLY(747) and N(756)ITY(759) motif of the beta(3) cytoplasmic tail and play the major role in the receptor activation. In this study, we investigated the role of the membrane distal region of human beta(3) cytoplasmic tail and specifically the N(743)NPLYKEA(750) and T(755)NITYRGT(762) sequence that contains an NXXY motif, in platelet aggregation, secretion, alpha(IIb)beta(3) activation (PAC-1 binding) and fibrinogen binding. We synthesized two peptides corresponding to the above sequences as well as their conjugates with the Tat(48-60) cell-penetrating peptide. The capability of conjugates to penetrate the platelet membrane was investigated with confocal laser scanning microscopy using carboxyfluorescein (CF)-labeled peptides. Our results showed that the conjugated with the Tat(48-60) sequence peptides penetrate the platelet membrane and inhibit platelet aggregation in both PRP and washed platelets in a dose-dependent manner. The Tat-beta(3)743-750 conjugate exhibited similar inhibitory activity in PRP and in washed platelets whereas the Tat-beta(3)755-762 conjugate was more potent inhibitor of aggregation in washed platelets than in PRP. Both conjugated peptides were also able to inhibit P-selectin membrane expression as well as PAC-1 and fibrinogen binding to the platelets, the Tat-beta(3)755-762 conjugate being more potent than Tat-beta(3)743-750. The Tat(48-60) peptide and the peptides beta(3)743-750 and beta(3)755-762, which were not conjugated to the Tat(48-60) sequence, did not exhibit any inhibitory effect on the above parameters. In conclusion, the present study shows for the first time that the peptide analogs of the intracellular domain of the beta(3) subunit beta(3)743-750 and beta(3)755-762 conjugated to the cell-penetrating peptide Tat(48-60) are capable of penetrating the platelet membrane and expressing biological activity by inhibiting the activation of alpha(IIb)beta(3), the fibrinogen binding to the activated receptor as well as platelet aggregation. Further studies are necessary to support whether such conjugated peptides may be useful tools for the development of potent antiplatelet agents acting intracellularly through the platelet integrin alpha(IIb)beta(3).

Spatial Coordination of Kindlin-2 with Talin Head Domain in Interaction with Integrin β Cytoplasmic Tails

“…Thus, the effects of ␤ 3 ⌬755, as well single point mutations at S752P or Y759A in the ␤ 3 CT on integrin activation (44 -49) can be attributed to inhibition of kindlin-2 binding. Also, the inhibition of integrin ␣ IIb ␤ 3 activation in platelets by importable peptides corresponding to ␤ 3 (743-750) and ␤ 3 (749 -756), which overlap the kindlin-2 binding core (50,51), can now be attributed to effects on kindlin binding. Expression of the ␤ 3 CT⌬748 and ␤ 3 CT (CORE) as a PSGL chimeric protein also suppressed integrin activation, suggesting that the membrane proximal and membrane distal regions of the ␤ 3 CT contribute independently to integrinmediated responses, observations consistent with the cooperativity between talin and kindlin-2 in integrin function.…”

Accessible Synthetic Probes for Staining Actin inside Platelets and Megakaryocytes by Employing Lifeact Peptide

“…This is also in agreement with studies in which myristoylation and palmitoylation were employed to enhance the activity of anti-platelet drugs targeting receptors at the inner leaflet of transmembrane proteins. [ 16c , 20 ]…”

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

“…To determine whether b etulinic acid chemotypes folds in the pharmacophore model of IP receptor agonists we determined its 3D structure. The 3D architecture of betulinic acid was build on the basis of the recently determined X-ray structure of 3β-Hydroxylup-20(29)-en-28-yl 1H-imidazole-1-carboxylate (23) and 2D 1 H- 1 H NOESY NMR data. As can be seen in Figure 4, in the NMR derived architecture, betulinic acid is composed by a carboxylate group that is located in a distance of 11.2 Å from the hydroxyl group and these two chemotypes are connected by a planar hydrophobic spacer composed by a tetracyclic ring system.…”

“…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. Platelets were then incubated with PAC-1-FITC and anti-CD62P-PE for 20 min in the dark at room temperature; diluted (1:5, v/v) with 10 mM PBS, pH 7.4 and immediately analyzed by flow cytometry (FACsCalibur, Becton-Dickinson, San Jose, CA, USA) as previously described (29). Platelets were gated according to staining for the platelet specific antigen, CD61.…”