A Ser⁷⁵²→Pro substitution in the cytoplasmic domain of β3 in a Glanzmann thrombasthenia variant fails to prevent interactions between the αIIbβ3 integrin and the platelet granule pool of fibrinogen

Abstract

Summary. A Glanzmann thrombasthenia variant with a b3Ser 752 fi Pro cytoplasmic domain substitution has platelets that fail to aggregate or bind soluble fibrinogen (Fg) after activation. Despite this, Fg is normally present in the a-granules. We have used immunoelectron microscopy to examine the reactivity of Fg with the different pools of aIIbb3 in the patient's platelets. Immunogold labelling was performed on cryosections using an anti-ligand-induced binding site (LIBS) monoclonal antibody (mAb), which binds to aIIbb3 only when Fg is bound, or a mixture of two anti-receptor-induced binding site (RIBS) mAbs that specifically recognize receptor-bound Fg. Labelling of the a-granule membrane and channels of the surface-connected canalicular system in unstimulated platelets confirmed that the mutated aIIbb3 retains the capacity to transport Fg.When the patient's platelets were stimulated with ADP in the presence of Fg, as expected there was a much-decreased activation of surface-exposed aIIbb3. However, thrombininduced activation was associated with both secretion and a rapid increase in the labelling of internal membrane systems by anti-RIBS and anti-LIBS mAbs, with mobilization of the internal Fg pool. Yet labelling on the surface of the patient's platelets was transient. Our studies implied that aIIbb3 in platelets may bind fibrinogen in different activation states and that this patient specifically lacked highaffinity binding.

An intracytoplasmic β3 Leu718 deletion in a patient with a novel platelet phenotype

“…Labeling of P-selectin in the a-granule membrane (including the giant granules) suggested a normal initial granule biogenesis ( Figure 2F). Fibrinogen was present in the a-granules, including the giant forms, and sometimes seemed to be in close association with the delimiting membrane, thus suggesting normal aIIbb3 cycling ( Figure 2G) (for images of control platelets, see Nurden et al 6 ). A morphometric analysis ( Figure 2H) confirmed platelet anisotrophy and altered a-granule concentration and size.…”

“…6,11,20,21 Neither patient exhibited MTP, which was present in a third family with a novel ITGB3 intronic mutation and frameshift that extended the b3 cytoplasmic domain by 40 amino acids and which locked aIIbb3 in a resting state by distancing the talin and kindlin-3 binding sites from the membrane. 22,23 Genetic variants affecting aIIb and b3 cytoplasmic domains that produce MTP show that the mutations result in an altered cytoskeletal architecture and reduced proplatelet formation.…”

Talin is required for integrin-mediated platelet function in hemostasis and thrombosis

“…The ability of talin-defi cient megakaryocytes to package fi brinogen into ␣ granules is noteworthy in light of the defect in activation of integrin ␣ IIb ␤ 3 in these platelets. It is possible that residual talin in early megakaryocytes may permit normal fi brinogen uptake; however, we have also observed normal platelet fi brinogen in the ␤ 3(L746A) platelets that have a similar defect in ␣ IIb ␤ 3 integrin activation (unpublished data), and human platelets with a ␤ 3(S752P) mutation also contain normal quantities of fibrinogen in spite of manifesting defective ␣ IIb ␤ 3 activation ( 27 ). Thus, even though integrin ␣ IIb ␤ 3 ligand binding function is required for normal fibrinogen uptake in megakaryocytes ( 26 ), activation of the integrin is not needed.…”

Glanzmann thrombasthenia: a review of ITGA2B and ITGB3 defects with emphasis on variants, phenotypic variability, and mouse models

“…Nevertheless, his platelets supported clot retraction and possessed a normal ␣-granule pool of Fg (Table 1). 41,42 A heterozygous Ser752Pro substitution in the ␤3 cytoplasmic tail (Figure 2) accounted for the defect and was said by the authors to be associated with a null allele on the basis that his daughter's platelets, which also expressed 50% ␣IIb␤3, aggregated normally but did not possess the ␤3 cytoplasmic domain mutation. 41 In modifying ␤3 intracytoplasmic domain structure (Figure 2), Pro752 not only abrogates ␣IIb␤3 and ␣v␤3 activation, it also blocks "outside-in signaling" and cell spreading after integrin engagement on surface-bound adhesive proteins.…”

“…41 In modifying ␤3 intracytoplasmic domain structure (Figure 2), Pro752 not only abrogates ␣IIb␤3 and ␣v␤3 activation, it also blocks "outside-in signaling" and cell spreading after integrin engagement on surface-bound adhesive proteins. [42][43][44][45] More precisely, the mutation prevents the binding to ␤3 of kindlin-3, a cytoplasmic protein required for integrin activation (for more information on kindlin-3, see "LAD-3 syndrome"). 46 A similar platelet phenotype was reported for a black American child shown to be compound heterozygote with a nonexpressed allele and platelets with intermediate amounts of ␣IIb␤3 containing a truncated ␤3 with only 8 membrane proximal amino acids of the 47 that compose the cytoplasmic tail and therefore lacking multiple active sites, including those responsible for talin binding.…”