A point mutation in the cysteine-rich domain of glycoprotein (GP) IIIa results in the expression of a GPIIb-IIIa (αIIbβ3) integrin receptor locked in a high-affinity state and a Glanzmann thrombasthenia–like phenotype

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Abstract
This article reports a Glanzmann thrombasthenia (GT) patient, N.M., with a point mutation in the third cysteine-rich repeat of beta3-integrin or platelet glycoprotein (GP) IIIa, leading to the expression of a constitutively activated fibrinogen receptor. The diagnosis of GT was based on a severely reduced platelet-aggregation response to a series of agonists and approximately 20% of surface-expressed GPIIb-IIIa. The patient's GPIIb-IIIa constitutively expressed epitopes recognized by antibodies to ligand-induced binding sites (LIBS) and also spontaneously bound the fibrinogen-mimetic antibody, PAC-1. Furthermore, significant amounts of bound fibrinogen were detected on his platelets ex vivo. No signs of platelet activation were observed on sections of unstimulated platelets from N.M. by electron microscopy. Immunogold labeling highlighted the presence of surface-bound fibrinogen but revealed platelet heterogeneity with regard to the surface density. When the patient's platelets were stimulated by thrombin-receptor activating peptide, amounts of surface-expressed GPIIb-IIIa increased and the aggregation response improved, although it failed to normalize. Platelets from N.M. were able to adhere and spread on immobilized fibrinogen. Sequence analysis of genomic DNA from N.M. revealed a homozygous g1776T>C mutation in GPIIIa, leading to a Cys560Arg amino acid substitution. A stable Chinese hamster ovary (CHO) cell line was prepared expressing surface GPIIb-Arg560IIIa. Like platelets from the patient, GPIIb-Arg560IIIa-transfected CHO cells constitutively bound LIBS antibodies and PAC-1. They also showed an enhanced ability to adhere on surface-bound fibrinogen. Overall, these data demonstrate that a gain-of-function mutation can still be associated with a thrombasthenic phenotype even though platelets show spontaneous fibrinogen binding.

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