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

Integrins are membrane receptors which mediate cell-cell or cell-matrix adhesion. Integrin alpha(IIb)beta3 (glycoprotein IIb-IIIa) acts as a fibrinogen receptor of platelets and mediates platelet aggregation. Platelet activation is required for alpha(IIb)beta3 to 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 alpha(IIb)beta3. However, isolated alpha(IIb)beta3 was 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 alpha(IIb)beta3 conformation such as the Arg-Gly-Asp-Ser peptide and alpha-chymotrypsin. These data suggested that a functional Arg-Gly-Asp binding site was present within alpha(IIb)beta3 and that the patient's defect was not secondary to a blockade of alpha(IIb)beta3 in a noncompetent conformational state. This was evocative of a defect in the coupling between platelet activation and alpha(IIb)beta3 up-regulation. We therefore sequenced the cytoplasmic domain of beta3, 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 alpha(IIb)beta3 from cellular activation because (i) it is not a polymorphism, (ii) it is the only mutation in the entire alpha(IIb)beta3 sequence, and (iii) genetic analysis of the family showed that absence of the Pro-752 beta3 allele was associated with the normal phenotype. Our data thus identify the C-terminal portion of the cytoplasmic domain of beta3 as an intrinsic element in the coupling between alpha(IIb)beta3 and platelet activation.