Glanzmann Thrombasthenia – IIb3 (Glycoprotein IIb/IIIa; CD41/CD61)

Definition and History

Glanzmann thrombasthenia is an inherited hemorrhagic disorder characterized by a severe reduction in, or absence of, platelet aggregation in response to multiple physiologic agonists because of qualitative or quantitative abnormalities of platelet glycoprotein IIb (GPIIb; CD41) and/or 3 (GPIIIa, CD61).1–4

In 1918, Eduard Glanzmann, a Swiss pediatrician, described a group of patients with hemorrhagic symptoms and a defect in platelet function, namely the ability to retract clots (“weak” platelets or thrombasthenia).5 Subsequent studies demonstrated that thrombasthenic patients have prolonged bleeding times and that platelets from thrombasthenic patients fail to aggregate in response to physiologic agonists such as adenosine diphosphate (ADP), epinephrine, collagen, and thrombin,6–9 and have markedly reduced6,8–10 levels of platelet fibrinogen. In the mid-1970s, Nurden and Caen11 and Phillips and colleagues12 discovered that thrombasthenic platelets are deficient in both IIb and 3. Later studies demonstrated that IIb and 3 form a calcium-dependent complex in the platelet membrane that functions as a receptor for fibrinogen and other adhesive glycoproteins.13–16 Cloning and sequencing of the complementary DNAs for IIb17 and 318 identified them as separate protein subunits that are members of the integrin receptor superfamily19 and permitted the molecular biologic characterization of patients with the disorder. Identification of the DNA defects in selected patients has provided information on the structure-function relationships of the IIb3 receptor and permitted DNA-based carrier detection and prenatal diagnosis (see database of Glanzmann patients and their molecular biologic defects at

Etiology and Pathogenesis

Glanzmann thrombasthenia is a rare disorder characterized by autosomal recessive inheritance with a worldwide distribution. In regions where consanguineous matings are common, groups of patients with the disorder have been identified, and in several populations founder mutations have been identified by analyzing polymorphisms in the DNA surrounding the affected mutation. These include 42 patients from South India; 39 patients from the Iraqi-Jewish population in Israel; 46 Arab patients from Israel, Jordan, and Saudi Arabia; 30 patients from Italy; and a smaller number of patients from 3 Gypsy families.10,20–27 Perhaps the highest frequency of a Glanzmann thrombasthenia mutation is found in the Iraqi-Jewish population where the most common mutation causing Glanzmann thrombasthenia was found in 6 of 700 individuals in that population.27

The platelet IIb3 receptor is required for platelet aggregation induced by all of the agonists thought to operate in vivo (ADP, epinephrine, thrombin, collagen, thromboxane A2; see Chap. 114).13–16 Consequently, abnormalities in the receptor result in a failure of platelet plug formation at sites of vascular injury, leading to excessive bleeding and bruising.

The IIb3 receptor is also responsible for the uptake of fibrinogen from plasma into platelet granules,28–31 hence, patients with Glanzmann thrombasthenia have markedly reduced levels of platelet fibrinogen.6,8,9,32,33 Clot retraction requires platelets with intact IIb3 receptors,34–36 presumably to make contact with fibrin, and thus, patients with Glanzmann thrombasthenia usually have abnormal clot retraction.6

Defects in either IIb or 3 result in the same functional defect because both subunits are required for receptor function (see Chap. 114). Biosynthetic studies indicate that IIb and 3 form a complex soon after protein synthesis in the rough endoplasmic reticulum37–39; subsequent posttranslational processing40 and transport to the platelet membrane require that the complex be intact (Fig. 121–2).41,42 Complex formation protects each of the glycoproteins from proteolytic digestion,37–40 so if either IIb or 3 is absent or unable to form a normal complex, the other subunit will be rapidly degraded. Thus, a deficiency in either glycoprotein produces a deficiency in both. Because complex formation and vesicular transport are also required for proteolytic processing of pro-IIb into its constituent IIb and IIb subunits,40 if complex formation and/or vesicular transport does not occur normally, the very small amount of residual IIb will be pro-IIb, not mature IIb.43 Pro-IIb has been reported to bind to the membrane-bound endoplasmic reticulum chaperone calnexin, providing a potential mechanism for assessing whether the protein has undergone proper folding (calnexin cycle) and perhaps explaining how the receptor adopts a bent configuration.44,45

3 (Glycoprotein [GP] IIIa) can alsocombine with the V-integrin (CD51) subunit to form the V3 “vitronectin receptor” (see Fig. 121–2 and Chap. 114).18,46,47 Despite its common name, this receptor can bind many of the same adhesive glycoproteins as IIb3, although there are some differences in ligand preference and binding sequences.47–51 A small number of V3 receptors are present on platelets (50–100 per platelet)50,52,53; osteoclasts, endothelial cells, macrophages, vascular smooth muscle, and uterine cells, among others, also have V3 receptors.54–56 In general, Glanzmann thrombasthenia patients with defects in 3 also are deficient in V3, whereas patients with defects in IIb have either normal or increased numbers of platelet V3 receptors.50,53,55,57–59 One exception to this rule is a patient with a defect in 3 (H280P) that interferes with IIb3 biogenesis to a much greater extent than V3 biogenesis.60 At present, there is no evidence that patients who lack V3 receptors in addition to lacking IIb3 receptors have a more severe hemorrhagic diathesis or suffer from any other abnormalities, perhaps because alternative receptors containing V associated with other subunits can substitute for V3.53 Upregulation of 21 on osteoclasts of Iraqi-Jewish patients with Glanzmann thrombasthenia has been reported as a potential compensatory mechanism to explain the lack of bone changes despite the deficiency in osteoclast V3.

The molecular biologic abnormalities in more than 100 patients with Glanzmann thrombasthenia have been identified60a and they are listed in an Internet database that is updated continuously61 ( Figure 121–3 contains information on mutations of particular interest. Of note, many of the patients with identified mutations are compound heterozygotes rather than homozygotes, indicating that a sizable number of silent carriers are present in the population. Where consanguinity is common, the disorder is more likely to be caused by a homozygous mutation arising in a founder, but even under these circumstances, more than one mutation may be present. Thus, in the Iraqi-Jewish population, in which consanguinity has been present from 586 BCE to the present, two separate mutations have been identified in more than one family.27 Most of the missense mutations result in decreased expression of IIb3 on the surface of platelets. This probably reflects the stringent structural requirements for proper folding and complex formation.

Mutations in IIb3 Within the Metal Ion-Dependent Adhesion Site of 3 (GPIIIa) and the Interface with the IIb (GPIIb) -Propeller

A metal coordination site or metal ion-dependent adhesion site (MIDAS) domain,62 which is highly conserved in six integrin receptor -chain subunits and required for ligand-binding,63 is also present in the A (or I-like) domain of the 3 subunit.64,65 Mutagenesis and molecular modeling experiments suggested that a highly conserved DxSxS amino acid sequence66 motif plus additional coordinating residues are brought together in the three-dimensional structure of the 3 subunit to form a cation-binding sphere of the MIDAS domain,62 and this was confirmed by the crystal structures of V3 and later IIb3 (see Figs. 114–3 and 121–3).67,68 Thus, the 3 MIDAS is composed of Asp119, Ser121, Ser123, Glu220, and Asp251. A region originally termed the ligand-associated metal-binding site (LIMBS) in V3,69 but now termed the synergy metal-binding site (SyMBS) in IIb3,65 binds a Ca2+ ion and is required for binding of ligands to the MIDAS. It is composed of atoms from D158, N215, D217, P219, and E220. 3 Residues 214 and 216 are in close proximity with both the SyMBS residues and the interface with IIb. Adjacent to the MIDAS domain is a metal ion site termed the ADMIDAS (adjacent to metal ion-dependent adhesion site), in which calcium is coordinated by Ser123, Asp126, Asp127, and Met335 in unliganded V3 and IIb3, but Asp251 substitutes for Met335 in the ligand bound structures of both V3 and IIb3. The crystal structures also demonstrated that peptide ligands containing the RGD cell adhesion sequence interact with IIb3 and V3 in part by coordination of the metal ion in the MIDAS by the aspartic acid in the RGD peptide.69,70 The low-molecular-weight drugs eptifibatide and tirofiban, which block ligand binding to IIb, have negatively charged regions that also interact with the MIDAS cation.68 The fibrinogen -chain C-terminal dodecapeptide mediates binding to IIb3 and a crystal structure of the complex demonstrates that an aspartic acid carboxyl oxygen coordinates the MIDAS cation whereas the carboxy-terminal valine interacts with the nearby cation in the ADMIDAS.68,70 A number of mutations in patients with Glanzmann thrombasthenia have been identified within the cation-binding sphere of the MIDAS domain (see Fig. 121–3). Two mutations D119Y (Cam variant)71 and D119N (patient NR),72 are located within the conserved DxSxS amino acid motif and produce severe abnormalities of ligand binding to IIb3, but do not affect IIb3 surface expression. Mutations at residues R214 and R216 result in abnormal IIb3 receptors that cannot bind ligand and are very sensitive to dissociation by calcium chelation, perhaps because they are at the IIb3 interface.20,73–75 Disrupting the SyMBS with a D217V mutation also leads to Glanzmann thrombasthenia despite the expression of normal amounts of IIb3.76 Further support for the importance of the MIDAS domain, SyMBS, and adjacent residues comes from studies in which the mutations D119N, R214W, D217N, E220Q, and E220K were introduced into Chinese hamster ovary (CHO) cells in vitro and shown to result in functional abnormalities.77

The interface between the IIb -propeller 3 also involves, in part, the interaction between 3 R261, contained in a four-amino-acid 310 helix, with a number of hydrophobic residues in the IIb -propeller arranged as inner and outer rings, making up a cage.67 A 3 L262Y mutation, adjacent to R261 results in disruption of the helix and an unstable IIb3 complex that is expressed on the surface of platelets but is unable to bind fibrinogen.78 The platelets of the patient with this mutation were able to bind fibrin and support clot retraction, suggesting different requirements for fibrinogen and fibrin binding.

Mutations in IIB3 Within the GPIIb (-Chain) -Propeller Sequence

Based on their homology to another integrin subunit, the amino-terminal 450 amino acids of IIb and the homologous region in V, which contain the minimal ligand-binding sequence,79 were predicted to fold into seven repeat (blade) -propellers, containing four cation-binding sites,80 and this prediction was confirmed by the crystal structures of both V and IIb.67,68 The upper surface of the propeller interacts with the 3 subunit -A (or I-like) domain to form the head of the IIb3 complex which is the site of ligand binding. Each repeat (blade) contains four strands that are connected by loops. The four calcium binding sites in IIb, which are in -hairpin structures, are located in loops on the undersurface of the propeller. Ligand binding in IIb has been localized to a hydrophobic (F160, Y190, F231) and negatively charged (D224) pocket that lies adjacent to the MIDAS domain in 3, and is composed of contributions from the loops that link blade 2 to blade 3 (residues 144–171), strand 2 to strand 3 in blade 3 (residues 186–193), and blade 3 to blade 4 (residues 223–236). IIb contains a unique “cap” subdomain made up of four insertions in propeller loops (residues 72–88, 111–126, 147–166, 200–217) that plays a ligand binding role similar to that of the I domains present in some integrin receptors.68

Glanzmann thrombasthenia missense mutations located within the IIb -propeller (see Fig. 121–3) primarily affect transport of the IIb3 complex to the cell surface,59,81–84 but several missense mutations and an insertion result in functionally defective receptors. Thus, Y143H affects soluble ligand binding but not adhesion or clot retraction,85 and P145A, which has been identified in several kindreds,20,86 and P145L, prevent ligand binding. A two-amino-acid insertion at residues 161 and 162, as well as a T176I missense mutation, also affect ligand binding.87–89 A L183P mutation, which is near to, but not in the loop containing Y190, affects both receptor expression and function.90

Mutations in IIb3 that Affect Receptor Activation

Several 3 missense mutations (C560R, V193M) result in the receptor adopting a high-affinity ligand binding state, which is paradoxical as it results in a bleeding diathesis.91,92 A 3 S527F mutation in the third I-EGF domain was also associated with a constitutively active receptor, presumably because it prevents the receptor from assuming a bent, inactive conformation.93 The cytoplasmic domain of 3 plays a functional role in integrin activation and the regulation of ligand binding.94–96 Two Glanzmann thrombasthenia mutations have been identified in this region. One is an R724X nonsense mutation (patient RM)97 that results in the deletion of the carboxy-terminal 39 residues of 3 and the other is a 3 S752P missense mutation (patient P or Paris I).94,96,98 This latter patient is unusual in that he had a generally mild history of excessive hemorrhage, but he did have a prolonged bleeding time and his platelets did not aggregate in response to ADP. These mutations do not severely affect surface expression of platelet IIb3 complexes, but both mutant receptors are unresponsive to agonist stimulation. Mammalian cell expression studies of these mutations show normal adhesion to immobilized fibrinogen, but abnormal cell spreading. Cells expressing the S752P mutant receptors have reduced focal adhesion plaque formation and cells expressing the R724X mutant receptors have undetectable tyrosine phosphorylation of focal adhesion kinase, pp125FAK. These mutations provide evidence for the role of the 3 cytoplasmic tail in inside-out signaling (i.e., platelet signals that lead to IIb3 adopting a high-affinity ligand-binding conformation) and outside-in signaling (i.e., signaling to the interior of the platelet as a result of IIb3 binding ligand;