A Gain of Function Mutation in aIIbb3 Results in a Glanzmann Thrombasthenia Phenotype

Abstract number: P2125

Vanhoorelbeke¹ K, Pareyn¹ I, Melchior² C, Plançon² S, Margue² C, Pradier³ O, Fondu⁴ P, Kieffer² N, Deckmyn¹ H

¹¹Laboratory for Thrombosis Research, IRC, KU Leuven Campus Kortrijk, Belgium ²²Laboratoire de Biologie et Physiologie Intégrée, Université du Luxembourg, Luxembourg ³³Hôpital Erasme, ULB, Brussels, Belgium ⁴⁴CHU Brugmann, ULB, Brussels, Belgium

Introduction:  When platelets are activated, the conformation of their aIIbb3 receptor changes allowing the symmetric molecule fibrinogen to bind which results in platelet aggregate formation. Glanzmann Thrombastenia (GT) is a bleeding disorder due to qualitative or quantitative abnormalities in aIIbb3. The aim of this study was to identify the genetic defect in the aIIb or b3 gene of a patient diagnosed as having a variant form of GT.

Results:  Patient L.M. suffered from mild bleeding problems. Platelet aggregation in the presence of weak agonists (e.g. collagen, ADP . . .) was impaired but was normal in the presence of the strong agonist TRAP. Patient platelets expressed normal levels of aIIbb3. Two heterozygous mutations were identified in the aIIb gene (R to W and L to M) and one heterozygous mutation in the b3 gene (S to F). The mutation in the b3 gene was not identified in the gene of the sister, who also has less bleeding problems. Five mutant aIIbb3-expressing CHO-cell lines were constructed: (1) the R to W (RW), (2) the L to M (LM), (3) the double mutant (RWLM), (4) the S to F (SF) and (5) the triple mutant (RWLMSF). Expression levels of RW, LM, RWLM and SF aIIbb3 were normal whereas expression of RWLMSF aIIbb3 was 3 times lower. Using anti-LIBS (ligand induced binding site) antibodies, an open conformation induced by the SF mutation was demonstrated. This was further confirmed by showing that, in contrast to WT-aIIbb3 or LM, RW and RWLM cells, SF and RWLMSF expressing CHO-cells did not need DTT to bind PAC-1 or to form aggregates in the presence of fibrinogen and CaCl₂.

Conclusion:  This is the first report on a GT-patient, with normal aIIbb3-levels, of which the phenotype is due to a gain-of-function mutation in the b3-gene that results in spontaneous binding of fibrinogen thereby saturating the aIIbb3 receptors. When platelets are activated, receptors are already occupied by fibrinogen and can no longer support platelet aggregation. A strong stimulus then may cause exposure of a–granule aIIbb3 that still can support aggregation.