Page 2070 - Williams Hematology ( PDFDrive )

2044 Part XII: Hemostasis and Thrombosis Chapter 120: Hereditary Qualitative Platelet Disorders 2045

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Ser , Asp , Asp , and Met in unliganded integrin α β and inte- result in functionally defective receptors. Thus, Y143H affects soluble
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grin α β , but Asp substitutes for Met in the ligand-bound struc- ligand binding but not adhesion or clot retraction, and P145A, which
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tures of both integrin α β and integrin α β . The crystal structures also has been identified in several kindreds, 32,95 and P145L, prevent ligand
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demonstrated that peptide ligands containing the Arg-Gly-Asp (RGD) binding. A two-amino-acid insertion at residues 161 and 162, as well
cell adhesion sequence interact with integrin α β and integrin α β as a T176I missense mutation, also affect ligand binding. 96–98 A L183P
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in part by coordination of the metal ion in the MIDAS by the aspartic mutation, which is near to, but not in the loop containing Y190, affects
acid in the RGD peptide. 77,79 The low-molecular-weight drugs eptifibat- both receptor expression and function. 99
ide and tirofiban, which block ligand binding to the α subunit, have Mutations in Integrin α β That Affect Receptor Activation
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negatively charged regions that also interact with the MIDAS cation. Several β subunit missense mutations (C560R, V193M) result in the
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The fibrinogen γ-chain C-terminal dodecapeptide mediates binding receptor adopting a high-affinity ligand binding state, which is par-
to integrin α β and a crystal structure of the complex demonstrates adoxical as it results in a bleeding diathesis. 100,101 A β subunit S527F
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that an aspartic acid carboxyl oxygen coordinates the MIDAS cation mutation in the third I-EGF domain was also associated with a con-
whereas the carboxy terminal valine interacts with the nearby cation stitutively active receptor, presumably because it prevents the recep-
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in the ADMIDAS. 76,79 A number of mutations in patients with GT tor from assuming a bent, inactive conformation. The cytoplasmic
have been identified within the cation-binding sphere of the MIDAS domain of the β subunit plays a functional role in integrin activation
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domain (see Fig. 120–3, and Chap. 112, Fig. 112–11). Two mutations and the regulation of ligand binding. 103,104 Two GT mutations have been
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D119Y (Cam variant) and D119N (patient NR), are located within identified in this region. One is an R724X nonsense mutation (patient
the conserved DxSxS amino acid motif and produce severe abnormal- RM) that results in the deletion of the carboxyterminal 39 residues
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ities of ligand binding to integrin α β , but do not affect its surface of integrin β and the other is a β subunit S752P missense mutation
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expression. Mutations at residues R214 and R216 result in abnormal (patient P or Paris I). 106–108 This latter patient is unusual in that he had a
integrin α β receptors that cannot bind ligand and are very sensitive generally mild history of excessive hemorrhage, but he did have a pro-
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to dissociation by calcium chelation, perhaps because they are at the longed bleeding time and his platelets did not aggregate in response
integrin α –β interface. 32,82–84 Disrupting the SyMBS with a D217V to ADP. These mutations do not severely affect surface expression of
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mutation also leads to GT despite the expression of normal amounts platelet integrin α β complexes, but both mutant receptors are unre-
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of the integrin protein. Further support for the importance of the sponsive to agonist stimulation. Mammalian cell expression studies of
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MIDAS domain, SyMBS, and adjacent residues comes from studies in these mutations show normal adhesion to immobilized fibrinogen, but
which the mutations D119N, R214W, D217N, E220Q, and E220K were abnormal cell spreading. Cells expressing the S752P mutant receptors
introduced into Chinese hamster ovary CHO cells in vitro and shown have reduced focal adhesion plaque formation and cells expressing the
to result in functional abnormalities. 86 R724X mutant receptors have undetectable tyrosine phosphorylation of
The interface between the α β-propeller and the β subunit also focal adhesion kinase, pp125 FAK . These mutations provide evidence for
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involves, in part, the interaction between β R261, contained in a four- the role of the β subunit cytoplasmic tail in inside-out signaling (i.e.,
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amino-acid 3 helix, with a number of hydrophobic residues in the α platelet signals that lead to integrin α β adopting a high-affinity lig-
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β-propeller arranged as inner and outer rings, making up a cage. A β and-binding conformation) and outside-in signaling (i.e., signaling to
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subunit L262Y mutation, adjacent to R261 results in disruption of the the interior of the platelet as a result of integrin α β binding ligand; see
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helix and an unstable integrin α β complex that is expressed on the Chap. 112, Figs. 112–3, 112–4, and 112–12).
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surface of platelets but is unable to bind fibrinogen. The platelets of Variants of Integrin α β in the Population The application
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the patient with this mutation were able to bind fibrin and support clot of missense variant whole-exome and whole-genome sequencing to
retraction, suggesting different requirements for fibrinogen and fibrin large numbers of individuals has provided valuable information on the
binding. frequency of missense variants in the general population and the fre-
Mutations in Integrin α β Within the α β–Propeller Sequence quency of the genetic alterations leading to GT. For the most part, the
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Based on their homology to another integrin α subunit, the aminoter- frequency of a variant in a population is a reflection of its impact on
minal 450 amino acids of integrin α and the homologous region in reproductive fitness and when it entered the population, with lower
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integrin α , which contain the minimal ligand-binding sequence, were frequencies for variants that entered the population more recently.
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predicted to fold into seven repeat (blade) β-propellers, containing four Thus, variants with minor allele frequencies (MAFs) of approximately
cation-binding sites, and this prediction was confirmed by the crystal 0.5 percent or less probably entered the population fewer than 2500 years
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structures of both α and α integrin subunits. 75,76 The upper surface of ago, when the recent explosive growth in human populations
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the propeller interacts with the β subunit β-A (or I-like) domain to form began. Data from a study 109A involving approximately 33,000 alle-
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the head of the integrin α β complex, which is the site of ligand binding. les from approximately 16,500 people demonstrated the presence
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Each repeat (blade) contains four β strands that are connected by loops. of 114 novel missense variant affecting approximately 10 percent
The four calcium-binding sites in the α subunit, which are in β-hairpin of the integrin α amino acids and approximately 9 percent of the
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structures, are located in loops on the undersurface of the propeller. Lig- β subunit amino acids. Thus, approximately 1.1 percent of the popula-
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and binding in integrin α has been localized to a hydrophobic (F160, tion studied carried at least one missense variant. None of the known
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Y190, F231) and negatively charged (D224) pocket that lies adjacent to GT mutations was observed in any of the alleles studied, indicating
the MIDAS domain in the β subunit, and is composed of contributions that they have MAFs of less than 0.01 percent and thus entered the
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from the loops that link blade 2 to blade 3 (residues 144 to 171), β strand population very recently. In fact, studies of two GT populations with
2 to β strand 3 in blade 3 (residues 186 to 193), and blade 3 to blade 4 (res- high intragroup marriages, Palestinian Arabs and French Manouche
idues 223 to 236). Integrin α contains a unique “cap” subdomain made gypsies, estimated that the GT mutations entered the population
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up of four insertions in β propeller loops (residues 72 to 88, 111 to 126, approximately 300 to 600 and approximately 300 to 400 years ago,
147 to 166, 200 to 217) that also plays a role in ligand binding. 76 respectively. 110,111 Several novel missense variants identified in this
GT missense mutations located within the integrin α β-propeller study affected one of the amino acids mutated in patients with GT, and
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(see Fig. 120–3) primarily affect transport of the integrin α β complex in two cases these variants were shown to profoundly affect expression
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to the cell surface, 68,90–93 but several missense mutations and an insertion of the receptor. In one case, a missense variant reduced expression by
Kaushansky_chapter 120_p2039-2072.indd 2045 9/21/15 2:20 PM