A Sequence within the Cytoplasmic Tail of GpIIb Independently Activates Platelet Aggregation and Thromboxane Synthesis

Abstract

All integrin ␣ subunits contain a highly conserved KXGFFKR motif in their cytoplasmic domains that plays a crucial role in the regulation of integrin affinity for their ligands. We show that a lipid-modified peptide corresponding to the cytoplasmic region, 989 -995, of the platelet integrin subunit glycoprotein GpIIb (␣IIb), palmitoyl-KVGFFKR (Ppep; 10 M), but not a similarly modified scrambled peptide (palmitoyl-FKFVRGK), can specifically induce platelet activation and aggregation equivalent to that of strong agonists such as thrombin. Ppep-induced aggregation is also associated with indices of platelet activation including thromboxane A 2 (TXA 2 ) synthesis (EC 50 ‫؍‬ 45 ؎ 5 M), secretion of ␣-granules detected as enhanced surface expression of P-selectin (EC 50 ‫؍‬ 52 ؎ 8 M), and conformational changes in GpIIb/IIIa measured by the monoclonal antibody, PAC-1 (EC 50 ‫؍‬ 3.7 ؎ 1 M). The TXA 2 receptor antagonist, SQ29548, PGE 1 , and the ADP scavenger, apyrase, differentially inhibit the aggregation response and TXA 2 synthesis in response to Ppep. Similarly, GpIIb/IIIa antagonists (RO-449883 and integrelin), which inhibit aggregation by greater than 90%, have little effect on peptide-induced TXA 2 synthesis, suggesting that this event is independent of fibrinogen binding to GpIIb/IIIa. Alanine-stepping of the Ppep sequence identifies GFFK(991-994) as the critical residues in all peptidemediated events. We conclude that this peptide can imitate the cytoplasmic domain of GpIIb and initiate parallel but independent signaling pathways, one leading to ligand binding and platelet aggregation and the other to intracellular signaling events such as TXA 2 synthesis and secretion.Integrins are a family of cell adhesion molecules composed of two subunits, ␣ and ␤, which form a complex on the cell surface. Ligand recognition by integrins may be modulated by intracellular signals that interact with the cytoplasmic tails of the subunits. This has been demonstrated most clearly for the platelet glycoprotein (Gp) 1 IIb/IIIa (␣IIb␤3), the most abundant platelet integrin, which acts as a receptor for fibrinogen, fibronectin, and other RGD-containing macromolecules (1). Under resting conditions, this receptor has a low affinity for its ligands (2). However, when platelets are stimulated by agonists such as thrombin or ADP, GpIIb/IIIa undergoes a conformational change (3) detected by the appearance of neoepitopes for monoclonal antibodies such as PAC-1 (4) and acquires a high affinity binding for its ligands, principally fibrinogen (5, 6). The binding of fibrinogen results in platelet aggregation, an early step in the generation of a thrombus. Deletion or mutation of the cytoplasmic domains of the integrin subunits can produce a constitutively active or inactive receptor (7-9), suggesting that signals resulting from cell activation interact with the intracellular components of GpIIb/IIIa to modify ligand recognition (inside-out signaling).The cytoplasmic domains are also important for events occurring as a consequence of ligand...

A structural basis for integrin activation by the cytoplasmic tail of the α IIb -subunit

“…However, the interpretation of such mutational data has remained conjectural, because the structure of the ␣ IIbcytoplasmic tail, as well as that of any of the ␣-subunits, is unknown. Recently, it has been shown that a lipid-modified peptide containing only the membrane-proximal region of ␣ IIb (K989-R995) can penetrate the platelet membrane and induce ␣ IIb ␤ 3 activation (8). This observation attests to the significance of the N-terminal region of the ␣ IIb -cytoplasmic tail in integrin activation and indicates that such peptides can adopt a functionally relevant conformation.…”

ICln, a Novel Integrin αIIbβ3-Associated Protein, Functionally Regulates Platelet Activation

“…Platelet Function-Gel-filtered platelets were prepared as previously described (1,4). Platelet aggregation assays were performed as described using 0.1 unit/ml thrombin or 10 M U46619, a thromboxane mimetic.…”

“…have previously established that the conserved amino acid sequence KVGFFKR, immediately adjacent to the transmembrane domain of the platelet ␣ IIb integrin subunit, plays a critical role in the regulation of integrin activation (4,42). To identify the protein-protein interactions responsible for this regulation, we synthesized a biotin-tagged peptide corresponding to this sequence and used it as a probe to identify high affinity protein interactions dependent on this motif.…”

“…Deletion or mutation of the highly conserved, membrane-adjacent KXGFFKR motif in ␣-integrins results in constitutive integrin activation (2,3). Furthermore, we and others have shown that addition of exogenous cell-permeable peptides corresponding to this sequence, KVGFFKR or KLGFFKR, directly activates integrins ␣ IIb ␤ 3 or ␣ 2 ␤ 1 , respectively (4,5). Thus this sequence plays a critical role in regulation of the activation state of the integrin.…”

Virtual Screening Using Combinatorial Cyclic Peptide Libraries Reveals Protein Interfaces Readily Targetable by Cyclic Peptides

“…There is also evidence that cyclic peptides may have improved membrane permeability, compared with linear peptides, in cases where the cyclic peptide can internally satisfy its own hydrogen bonds. 15 Other approaches that have been used to successfully transport linear peptides inside cell membranes, such as palmytoylation, 16 may also be used with cyclic peptides.…”