Ser-752 → Pro Mutation in the Cytoplasmic Domain of Integrin β3Subunit and Defective Activation of Platelet Integrin αIIbβ3(Glycoprotein IIb-IIIa) in a Variant of Glanzmann Thrombasthenia

Integrins are membrane receptors which mediate cell-cell or cell-matrix adhesion. Integrin αIIbβ3(glycoprotein IIb-IIIa) acts as a fibrinogen receptor of platelets and mediates platelet aggregation. Platelet activation is required for αIIbβ3to shift from noncompetent to competent for binding soluble fibrinogen. The steps involved in this transition are poorly understood. We have studied a variant of Glanzmann thrombasthenia, a congenital bleeding disorder characterized by absence of platelet aggregation and fibrinogen binding. The patient's platelets did not bind fibrinogen after platelet activation by ADP or thrombin, though his platelets contained αIIbβ3. However, isolated αIIbβ3was able to bind to an Arg-Gly-Asp-Ser affinity column, and binding of soluble fibrinogen to the patient's platelets could be triggered by modulators of αIIbβ3conformation such as the Arg-Gly-Asp-Ser peptide and α-chymotrypsin. These data suggested that a functional Arg-Gly-Asp binding site was present within αIIbβ3and that the patient's defect was not secondary to a blockade of αIIbβ3in a noncompetent conformational state. This was evocative of a defect in the coupling between platelet activation and αIIbβ3up-regulation. We therefore sequenced the cytoplasmic domain of β3, following polymerase chain reaction (PCR) on platelet RNA, and found a T → C mutation at nucleotide 2259, corresponding to a Ser-752 → Pro substitution. This mutation is likely to be responsible for the uncoupling of αIIbβ3from cellular activation because (i) it is not a polymorphism, (ii) it is the only mutation in the entire αIIbβ3sequence, and (iii) genetic analysis of the family showed that absence of the Pro-752 β3allele was associated with the normal phenotype. Our data thus identify the C-terminal portion of the cytoplasmic domain of β3as an intrinsic element in the coupling between αIIbβ3and platelet activation.