Διερεύνηση της συμβολής περιοχών της υπομονάδας β3 του υποδοχέα αΙΙbβ3 στη διαδικασία συσσώρευσης των ανθρώπινων αιμοπεταλίων (Doctoral thesis)
Σταθόπουλος, Παναγιώτης Τ.
Ρlatelets play a significant role in the pathogenesis of acute coronary syndromes. Among all glycoproteins-receptors, integrin αIIbβ3 has gained the interest of investigators, since it plays the central role in the final step of platelet aggregation and thrombus formation. On unstimulated platelets, the platelet receptor αIIbβ3 is presented in a closed conformation that prevents ligand binding. Upon platelet activation by several agonists, αIIbβ3 receives intracellular signals (inside-out signaling) that allow the cytoplasmic proteins, such as talin, β3-endonexin, Calciumand- Integrin Binding Protein, to interact with the cytoplasmic domains of αIIbβ3 subunits, resulting in platelet aggregation. The aim of this work was to develop anti-platelet agents able to inhibit thrombus formation by specifically disrupting the inside-out signalling pathway, using synthetic peptides based on the cytoplasmic region of β3 subunit. Peptide analogues derived from β3743-762 region (N743NPLYKEA750, N743NPLY(PO3H2)KEA750, T755NITYRGT762, E749ATSTFTN756, E749ATSTFTNITYRGT762 and N743NPLYKEA TSTFTN756) were synthesized in their free, palmitoylated and/or tagged with the Tat(G48RKKRRQRRRPPQ60) sequence and carboxyfluorescein(CF)-labeled in order to investigate their membrane permeability, as well as their inhibition potency on the platelet aggregation. Peptides were synthesized manually by solid-phase peptide synthesis on a Wang and/or Rink Amide resins, using the Fmoc/tBu protection strategy. The fluorescein- labeled peptides were generated by coupling N-Hydroxysuccinimide fluorescein to the amino-terminal end of the resin-bound peptide, prior to its cleavage and deprotection. Palmitoylated (Pal) and carboxyfluorescein(CF) -labeled analogues were prepared by acylation of the α- and ε-amino groups of the N-terminal lysine residue by palmitic anhydrite and NHS-fluorescein respectively. The conclusions of this work can be summarized as follows: # The conditions for the first amino acid attachment on Wang resin strongly affect the formation of byproducts, due to dipeptide formation. # Care must be taken during the Nα-Fmoc deprotection step of the second, from the C-terminal end, amino acid residue, in order to avoid the formation of diketopiperazine which lead to peptide sequences without the C-terminal dipeptide. # The cleavage of the peptides from the Rink Amide resin, under acidic conditions, leads to C-terminal N-alkylated peptide amides, independently of the peptide sequences, due to the Rink Amide linker decomposition. The formation of these byproducts can be prevented using as cleavage mixture the cocktail: 92.5% TFA, 5% DMB, 2.5% TIS (Reagent I).
|Institution and School/Department of submitter:||Πανεπιστήμιο Ιωαννίνων Σχολή Θετικών Επιστημών Τμήμα Χημείας|
|Keywords:||Φάρμακα, αντιαιμοπεταλιακά,Αποικοδόμηση rink amide βραχίονα,Πεπτίδια διαμεμβρανική μεταφορά,Wang ρητίνη, συνθετικά προβλήματα,Πεπτίδια, σύνθεση με fuoc / tbu στρατηγική,Αντιθρομβωτική δράση β3 πεπτιδικών αναλόγων|
|Appears in Collections:||Διδακτορικές Διατριβές|
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