Ser-752-->Pro mutation in the cytoplasmic domain of integrin beta 3 subunit and defective activation of platelet integrin alpha IIb beta 3 (glycoprotein IIb-IIIa) in a variant of Glanzmann thrombasthenia.

Abstract

Medical SciencesSer-752 -> Pro mutation in the cytoplasmic domain of integrin .83 subunit and defective activation of platelet integrin aIlbP3 (glycoprotein lib-Illa) in a variant of Glanzmann thrombasthenia (molcular genetlcs/flbnnogen receptor/Arg-Gly-Asp/poyerase chan reacdon) YI-PING CHEN*, ISABELLE DJAFFAR*, DOMINIQUE PIDARDt, BEAT (8,9), as well as for aLI32 (10). At least in the case of amb43 activation, modulation of affinity for its ligands is coordinated with conformational changes of the integrin (7,11). What remains obscure is the exact link between intracellular signals and aIIbP3 conformation modulation.Glanzmann thrombasthenia is a hereditary bleeding disorder characterized by the absence of platelet aggregation due to either an absence or a functional alteration of the integrin aII03 (12). In several cases, the genetic basis for the quantitative defect was found to be associated with alterations within the genes of ajib,83 subunits-i.e., either aib (glycoprotein lIb) (13,14) or 83 (glycoprotein IIa) (14, 15). In addition, the qualitative defects in aIlbm3 found in variants of Glanzmann thrombasthenia have also become the focus of investigations, and two mutations in Glanzmann variants that disrupted ligand binding sites of (3 have been characterized (16-18). Here we report a variant of Glanzmann thrombasthenia whose biochemical and genetic analyses are consistent with the idea that the functional defect is due to replacement of serine by proline in position 752 in the cytoplasmic domain of P3 and that this mutation impairs the coupling between cellular activation and up-regulation of arnb,83 affinity for fibrinogen. To our knowledge this is the first point mutation reported affecting integrin activation. METHODSPatient's Case. Patient P.'s case of variant Glanzmann thrombasthenia has been previously reported (19). Briefly, patient P. exhibited a life-long bleeding tendency with failure of platelets to aggregate and to bind fibrinogen in response to agonists such as ADP, collagen, or thrombin. The patient's platelets could support clot retraction. The presence of alb/3 in the patient's platelets was established by immunocytochemistry.Protein Analysis and Monoclonal Antibody Binding. Blood was collected in the presence of 5 mM EDTA, and platelets were prepared by conventional methods (20). Platelet proteins were solubilized in 2% NaDodSO4 and two-dimensional nonreduced/reduced NaDodSO4/PAGE was performed as described (21). Total platelet proteins (200 ,ug) were solubilized in 2% NaDodSO4 and separated in a first dimension in a 6% polyacrylamide tube gel. Disulfide bonds were reduced by soaking the gel in 10%6 (vol/vol) 2-mercaptoethanol, and the proteins were then separated again in a perpendicular direction in a 7-12% polyacrylamide slab gel. Proteins were detected by staining with Coomassie blue-R.The monoclonal antibody AP-2, which is specific for glycoprotein Ilb-IIla (22), the a11b13 integrin (a generous gift Abbreviations: RGDS, Arg-Gly-Asp-Ser; nt, nucleotide(s). lTo whom reprint requests...

Effects of Limiting Extension at the αIIb Genu on Ligand Binding to Integrin αIIbβ3

“…It is of interest to compare our data on the clamped receptor with observations on the platelets of a patient with Glanzmann thrombasthenia who had intact ␣IIb␤3 ectodomains but whose receptors were insensitive to inside-out signaling as a result of a mutation in the ␤3 cytoplasmic domain (26,36). The patient's receptors were able to support platelet adhesion to immobilized fibrinogen but not soluble fibrinogen binding in response to agonist stimulation.…”

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

“…The point mutation Ser7523 Pro in the ␤ 3 cytoplasmic tail was associated with an apparent ␣ IIb ␤ 3 activation defect in a patient with Glanzmann's thrombasthenia. 10 Prolonged inhibition of Ser/Thr phosphatases by calyculin A increased the phosphorylation of Thr753, which was accompanied by a decrease in platelet adhesion and spreading on surface-coated fibrinogen. 42 In an attempt to compete with the native integrin subunit for regulatory signals, peptides that mimic different, but partially overlapping, domains of the ␤ 3 subunit were designed.…”

“…Patients with Glanzmann's thrombasthenia, a severe bleeding disorder, have platelets that either lack ␣ IIb ␤ 3 (type I) or contain a defective ␣ IIb ␤ 3 that is unable to expose ligand binding sites (type III). 10 The Ser7523 Pro mutation in the ␤ 3 subunit of type III patients is of particular interest because it results in the loss of ligand binding. 11 Platelet agonists trigger inside-out signaling to ␣ IIb ␤ 3 via protein tyrosine kinases (PTKs) as herbimycin A, tyrphostin A47, and geldanamycin inhibit ligand binding.…”

The MIG-2/Integrin Interaction Strengthens Cell-Matrix Adhesion and Modulates Cell Motility

“…2, A and C). Previous studies have shown that a proline substitution mutation at Ser 752 , which is found in a variant of Glanzmann thrombasthenia, suppresses ␣IIb␤3 integrin activation (49,50). The S752P point mutation does not impair talin-H binding (28).…”