Inhibition of Platelet Integrin α _IIb β ₃ by Peptides That Interfere With Protein Kinases and the β ₃ Tail

Abstract

Abstract-␣-Thrombin stimulation of human platelets initiates inside-out signaling to integrin ␣ IIb ␤ 3 (glycoprotein IIb/IIIa), resulting in the exposure of ligand binding sites. In the present study, the regulation of ␣ IIb ␤ 3 via protein kinases was investigated in platelets permeabilized with streptolysin O by introducing peptides that interfere with these enzymes and with possible regulatory domains in the cytosolic tail of the ␤ 3 subunit. Compared with intact platelets, the permeabilized platelets preserved Ͼ80% of the aggregation, secretion, and ␣ IIb ␤ 3 ligand binding capacity. The peptide YIYGSFK, a substrate for Src kinases, inhibited ␣-thrombin-induced ligand binding to ␣ IIb ␤ 3 , but a reversed peptide with Y3 F substitutions (KFSGFIF) had no effect. Ligand binding to ␣ IIb ␤ 3 was also inhibited by the peptide RKRCLRRL, which binds irreversibly to the catalytic domain of protein kinase C. Peptides corresponding to parts of the protein C inhibitor and ␤ 2 -glycoprotein I were used as negative controls and failed to interfere with ligand binding. Possible target domains for protein kinases are present in the cytoplasmic tail of the ␤ 3 subunit. The LLITIHDR peptide, matching the membrane-proximal domain of ␤ 3 (residues 717 to 724), had no effect, but NNPLYKEA (residues 743 to 750), EATSTFTN (residues 749 to 756), and TNITYRGT (residues 755 to 762), which mimicked overlapping domains of the carboxy-terminal part of ␤ 3 , reduced ␣-thrombin-induced ligand binding by 60Ϯ4%, 97Ϯ1%, and 97Ϯ2% (nϭ3) at 500 mol/L peptide, respectively. These observations indicate that Src kinases and protein kinase C take part in inside-out signaling to integrin ␣ IIb ␤ 3 and identify target domains in ␤ 3 that contribute to the regulation of this integrin. Key Words: integrin ␣ IIb ␤ 3 Ⅲ glycoprotein IIb/IIIa Ⅲ protein kinase C Ⅲ protein tyrosine kinase Ⅲ platelets I ntegrins are heterodimeric cell surface receptors consisting of noncovalently associated ␣ and ␤ subunits. 1 Each subunit contains a large extracellular domain, a transmembrane domain, and a relatively short cytoplasmic tail (typically Ͻ70 residues for each subunit). Integrins function in a variety of biological processes, such as the differentiation, growth, and migration of cells, and in inflammation and wound healing. Many integrins are subject to intracellular modulation of their activation state for ligands, a process known as inside-out signaling. [2][3][4] An increase in binding affinity and subsequent ligand binding generates signals into the cell, a process known as outside-in signaling. [5][6][7][8] The regulation of platelet integrin ␣ IIb ␤ 3 is a good illustration of the significance of the rapid affinity regulation of integrins. Unstimulated platelets express ␣ IIb ␤ 3 in a conformation inaccessible to ligands, thereby preventing plateletplatelet interaction. On activation, inside-out signaling converts the integrin to a functional receptor for fibrinogen, fibronectin, von Willebrand factor, and vitronectin. 9 Fibrinogen binding to ␣ IIb ␤ 3 ...

Disruption of the Mouse μ-Calpain Gene Reveals an Essential Role in Platelet Function

“…Studies with pharmacological inhibitors of tyrosine kinases have shown that the platelet agonists trigger inside-out signaling via ␣IIb␤3 integrin by tyrosine phosphorylation of proteins in the molecular mass range of 54, 60, 64, 75, and 130 to 140 kDa (6,7,13,14). Moreover, the cytoplasmic domain of ␤3 integrin is itself tyrosine phosphorylated as a consequence of outside-in signaling and plays an important role in the regulation of integrin-myosin interaction during clot retraction (18,22).…”

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.…”

Direct Demonstration of Involvement of Protein Kinase Cα in the Ca2+-induced Platelet Aggregation

“…In the research of platelet granule secretion, several semi-intact assays have been established (4 -8, 19, 20). However, for platelet aggregation, only a few semi-intact aggregation assays have been established (8), and as far as we know, no stable assays with cytosol dependence have been established. at 4°C for 6 h, and the supernatant after removing the beads was examined by immunoblotting using another PKC␣-specific antibody (Sigma) and anti-pan-PKC␤, anti-PKC␦, and anti-PKC antibodies as described under "Experimental Procedures."…”

“…To overcome this difficulty, semi-intact assays using permeabilized platelets have been established for the study of granule secretion (4 -8). Although some semi-intact aggregation assays have now been developed, it has been difficult to demonstrate a cytosol dependence in these experiments (8).…”