α_IIbβ₃ expression is limited primarily to cells of the megakaryocytic lineage. However, the β₃ subunit is widely expressed as the beta subunit of the integrin α_vβ₃. Therefore, restriction of α_IIbβ₃ expression is limited by the promoter activity of α_IIb in cell types other than megakaryocytes.^20,21 Only a few exceptions have been reported. For example, α_IIbβ₃ is detected in certain cancer cells.²² α_IIbβ₃ has also been detected on early hematopoietic progenitors and on mast cells where it plays a role in adhesion and in the response of the latter cells to chronic inflammation.^23,24 Efficient surface expression of the α_IIbβ₃ complex requires α_IIb and β₃ in a ratio of 1:1.^25,26,27 Excess expression of one results in the accumulation of the excess in the endoplasmic reticulum where association of nascent α_IIb and β₃ subunits occurs. Indeed, a subset of patients with GT results from mutations that alter the biosynthesis of only one subunit. In turn, this impairs the surface expression of the complete α_IIbβ₃ complex.^28,29

α_IIbβ₃ is the most abundant protein on the platelet surface: there are approximately 80,000 copies on the surface of unactivated platelets.³⁰ An intracellular pool of α_IIbβ₃, located in the membrane of α-granules, can be translocated to the cell surface following platelet activation, increasing the amount of α_IIbβ₃ by 30% to 50%.^30,31,32 This high level of expression is important for efficient platelet function as platelets need to strongly adhere to multiple adhesive proteins to form a stable clot.

β₃ is expressed as a single polypeptide of 762 amino acid that folds into 10 distinct domains: a plexin-semaphorin-integrin (PSI) domain at the N-terminal, a βA domain that is inserted into the hybrid domain, followed by hybrid, EGF-1, EGF-2, EGF-3, EGF4, and βTD domains, a transmembrane domain (TMD) embedded in the lipid bilayer and a cytoplasmic domain^33,34 (FIGURE 26B.1). Folding is facilitated by 28 disulfide bonds formed from 56 cysteine residues. The β₃ βA domain has three divalent cation-binding sites: a metal ion-dependent adhesion site (MIDAS), a site adjacent to MIDAS, and a ligandinduced metal binding site, also referred to as the synergistic metal ion binding site. The β₃ A-domain assumes a nucleotide binding (or Rossmann) fold in which a central six stranded β-sheet is surrounded by eight α-helices^33,34 (FIGURES 26B.1, 26B.2 and 26B.3).

α_IIb has 1,008 amino acid and folds into six domains: β-propeller, thigh, calf-1, calf-2, transmembrane, and cytoplasmic domains.^33,34 α_IIb is initially synthesized as a single polypeptide chain. During protein folding and maturation, it is proteolytically cleaved at a site in the calf-2 domain into heavy and light chains by a furin-like enzyme in the Golgi complex. However, the heavy and light chains remain covalently associated via a disulfide bond.³⁵ Thus, in nonreduced sodium dodecyl sulfate gels, α_IIb has an apparent molecular weight of 130 kDa. In reducing gels, however, it consists of a 115 kDa fragment containing most of its extracellular domains and a 25 kDa fragment that contains part of the calf-2 domain, as well as the transmembrane and the cytoplasmic domains. At its N-terminus, α_IIb consists of seven β sheets containing approximately 60 amino acids each that fold into a seven-bladed β-propeller (FIGURE 26B.2A). Four divalent cation binding sites are found at the base of the
propeller.^33,34 A flexible loop between the thigh and the calf-1 domain (often referred to as the “genu” region) then allows the molecule to undergo a functionally important global structural rearrangement^33,34,36,37 (FIGURE 26B.1).

α_IIb and β₃ associate to form a noncovalent heterodimer. Several interfaces hold the heterodimer together: a large interface between the β-propeller and βA domain that buries approximately 1,600 Ų of surface area and with Arg261 of the βA domain protruding off-center into the β-propeller’s channel^33,34,38 (FIGURE 26B.2B); an interaction between the α_IIb and β₃ TMDs; and an interaction between the membrane proximal (MP) portions of each cytoplasmic domain.^39,40 Divalent cations are involved in this association because exposure to ethylenediaminetetraacetic acid causes the heterodimer to dissociate.^4,41 Although no divalent cation is observed at the α and β interface, cations in the β-propeller and thigh-calf-1 interface might allosterically contribute to α and β interactions.³³ The Arg-Gly-Asp or RGD motif present in many integrin ligands inserts into a crevice between the βA and β propeller domains. The Arg side chain inserts into a narrow groove on the top of the β-propeller, and the side chain of Asp protrudes into a cleft in the βA domain. The Gly residue lies at the interface between the α and β subunits where it makes several hydrophobic contacts. Overall, 355 Ų of surface area or 45% of the total surface area of the peptide is buried, thereby contributing to a stable ligand-receptor interaction⁴² (FIGURE 26B.3).

Structures of the α_IIbβ₃ TMD heterodimer, determined by nuclear magnetic resonance (NMR) spectroscopy, revealed that
the α_IIb and β₃ TMD pack together at two main interfaces with a vertical α_IIb TMD and tilted β₃ TMD.³⁹ At the outer membrane region, α_IIb and β₃ tightly pack against each other via a groove formed in the α_IIb G⁹⁷²XXXG⁹⁷⁶ motif and β₃ G⁷⁰⁸ (termed outer membrane clasp or OMC). The β₃ TMD forms a continuous helix that extends to the MP region of the cytoplasmic domain. A hydrophobic patch in the membrane-proximal region of β₃ is inserted into the membrane as a part of the TMD. Consequently, the β₃ TMD is long and crosses the lipid bilayer at an angle of 25 degrees. In contrast, the α_IIb TMD is a short and straight helix that ends at Gly991. Distal to Gly991, α_IIb makes a sharp turn toward β₃, enabling Phe992 and Phe993 to embed in the membrane and pack against β₃ residues Trp715 and Ile719. In addition, this places α_IIb Arg995 in close apposition to Asp723 of β₃, enabling an electrostatic interaction between them. The Phe packing interfaces and Arg995-Asp723 interaction completes the inner membrane interface (termed inner membrane clasp, or IMC)³⁹ (FIGURE 26B.4).

The combination of crystal and NMR structures and mutational studies has provided a detailed picture of integrin regulation. Integrin TMDs have two main interaction interfaces with three main contacts.^39,40 Mutations that disrupt these interactions favor the active form of the integrin. Charge reversal mutations of either α_IIb Arg995 or β₃ Asp723 disrupt the IMC and favor integrin activation, whereas combined charge reversals shift the equilibrium toward the inactive form.⁴³ Similarly, Phe to Ala mutations in the α_IIb MP region disrupt the IMC, inducing constitutive α_IIbβ₃ activity.⁴³

Activating mutations are also found in the OMC. Most are substitutions of the Gly residues in both the α_IIb G⁹⁷²XXXG⁹⁷⁶ motif and β₃ Gly708 with residues containing bulky side chain, such as Leu, Ile, or Asn.^44,45,46 The side chains of these residues disrupt the tight packing interface between α_IIb G⁹⁷²XXXG⁹⁷⁶ motif and the β₃ TMD.^39,47 Furthermore, the 25-degree tilting angle of the β₃ helix is required to enable simultaneous interactions at the IMC and OMC.³⁹ Mutations that change the β₃ tilting angle, such as Lys716 to Ala, also result in disruption of the association of the β₃ TMD with α_IIb.¹⁷ In short, integrins are kept in an inactive form by TM and cytoplasmic domain interactions. Following inside-out signaling, the α_IIb and β₃ TMD rearrange, causing integrin activation.

Integrin extracellular domains have multiple conformations, including those associated with inactive, active, and active ligand-occupied forms. The transition from one conformational state to another results in characteristic exposure of epitopes. A large inventory of conformation-specific α_IIbβ₃ antibodies has accumulated to study the relationship between α_IIbβ₃ conformation and function. How do the TMD and cytoplasmic domain rearrangements propagate to the integrin head
piece to cause higher ligand binding affinities? One hypothesis proposes that a bent integrin conformation has a low affinity for ligand, while a more open and extended conformation is associated with high ligand affinity³⁷ (FIGURE 26B.5). It has also been proposed that TMD and cytoplasmic domain separation results in extension of integrin extracellular domain at the genu region and swing out of the β₃ hybrid domain.⁴⁸ The β₃ hybrid domain movement is coupled through the βA α7-helix to rearrange the ligand binding crevice at the interface between β-propeller and βA domains.³⁸ This global structural rearrangement increases the affinity of α_IIbβ₃ for its ligand³⁷ (FIGURE 26B.5). An alternative hypothesis posits that the bent conformation can be an active conformer, and that extension of the molecule is a post-ligandbinding event. This theory also has experimental support.^33,42,49,50 This is an area where there is likely to be continued debate.