Activation of Platelet αIIbβ3 by an Exogenous Peptide Corresponding to the Transmembrane Domain of αIIb

Hang Yin,Rustem I Litvinov,Gaston Vilaire,Hua Zhu,Wei Li,Gregory A Caputo,David T Moore,James D Lear,John W Weisel,William F Degrado,Joel S Bennett

doi:10.1074/jbc.m605877200

Hang Yin, Rustem I Litvinov + Show 9 more

Open Access

https://doi.org/10.1074/jbc.m605877200

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Abstract

A transmembrane domain heterodimer, acting in concert with a membrane-proximal cytoplasmic domain clasp, is thought to maintain integrins in a low affinity state. To test whether helix-helix interactions between the alphaIIb and beta3 transmembrane domains regulate the activity of integrin alphaIIbbeta3, we synthesized a soluble peptide corresponding to the alphaIIb transmembrane domain, designated alphaIIb-TM, and we studied its ability to affect alphaIIbbeta3 activity in human platelets. alphaIIb-TM was alpha-helical in detergent micelles and phospholipid vesicles, readily inserted into membrane bilayers, bound to intact purified alphaIIbbeta3, and specifically associated with the transmembrane domain of alphaIIb, rather than the transmembrane domains of beta3, alpha2, and beta1, other integrin subunits present in platelets. When added to suspensions of gel-filtered platelets, alphaIIb-TM rapidly induced platelet aggregation that was not inhibited by preincubating platelets with the prostaglandin E(1) or the ADP scavenger apyrase but was prevented by the divalent cation chelator EDTA. Furthermore, alphaIIb-TM induced fibrinogen binding to platelets but not the binding of osteopontin, a specific ligand for platelet alphavbeta3. The peptide also induced fibrinogen binding to recombinant alphaIIbbeta3 expressed by Chinese hamster ovary cells, confirming that its effect was independent of platelet signal transduction. Finally, transmission electron microscopy of purified alphaIIbbeta3 revealed that alphaIIb-TM shifted the integrin from a closed configuration with its stalks touching to an open configuration with separated stalks. These observations demonstrate that transmembrane domain interactions regulate integrin function in situ and that it is possible to target intra-membranous protein-protein interactions in a way that can have functional consequences.

Highlights

Data supporting the heteromeric association of the ␣IIb and ␤3 TM domains in unstimulated platelets are largely indirect [2, 3]
It is noteworthy that mutations that either enhance or disrupt homomeric ␣IIb and ␤3 TM domain interactions in vitro can activate the intact integrin expressed in Chinese hamster ovary (CHO) cells
We show here that a synthetic peptide, designated ␣IIb-TM, corresponding to the wild type ␣IIb-TM domain, undergoes specific association with the ␣IIb-TM domain in micelles and bacterial ide; fluorescence activated cell sorting (FACS), fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; OPN, osteopontin; PGE1, prostaglandin El; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPG, 1-palmitoyl-2-oleoyl-sn-glycero-3-[phosphorac-(1-glycerol)]; Transmission Electron Microscopy (TEM), transmission electron microscopy; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; MOPS, 3-(N-morpholino)propanesulfonic acid

Summary

EXPERIMENTAL PROCEDURES

Peptide Synthesis and Purification—Peptides were synthesized using a 430A peptide synthesizer (Applied Biosystems) at 0.25-mmol scales and on a Rink amide AM resin (200 – 400 mesh) (Novabiochem) with a substitution level of 0.71 mmol/g. Anisotropy measurements were recorded by titrating a 64 nM FITC-␣IIb-TM peptide solution with increasing concentrations of purified ␣IIb␤3 protein. When the chimera contained the ␤1 TM domain, and there The ␣IIb-TM Peptide Induces Platelet Aggregation by Directly were nonsignificant increases in CAT synthesis when chimeras Activating ␣IIb␤3—The ability of ␣IIb-TM to interact with the. Cells—The ability of ␣IIb-TM to activate ␣IIb␤3 independent of signal transduction was corroborated by measuring fibrinogen binding to recombinant ␣IIb␤3 expressed by CHO cells using laser tweezers-based force spectroscopy [28]. This result suggests that ␣IIb-TM is able to induce an active ␣IIb␤3 conformation, oligomerization of the active molecules cannot occur because the required oligomerization site on ␣IIb is occupied by the peptide

DISCUSSION

Findings

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