Sizing up platelet defects

In this issue of Blood, Ghevaert and colleagues show that a mutation in the cytoplasmic domain of integrin β₃ is associated with dominantly inherited macrothrombocytopenia. This mutation is predicted to disrupt a conserved salt bridge with integrin α_IIb, and constitutively activates the integrin.

Large platelets are often encountered in the evaluation of thrombocytopenias, but most often these are acquired conditions. A rather long list of inherited thrombocytopenias is now appreciated.¹  Among the macrothrombocytopenias, the most commonly responsible genetic variations are heterozygosity for mutations in platelet glycoprotein Ibα and the Q43P polymorphism of β1 tubulin. The molecular regulation of platelet size is poorly understood, but circumstantial evidence supports a role for the platelet cytoskeleton and microtubular system. The lion's share of this evidence stems from the study of genetic defects in inherited disorders of platelet size. Although these disorders are quite uncommon, an understanding of the molecular basis of platelet size is important because subjects with abnormally high platelet volumes have enhanced platelet reactivity and are at risk for recurrent ischemic coronary syndromes.² 

Ghevaert and colleagues performed a thorough series of studies characterizing a pedigree for macrothrombocytopenia, with the mean platelet volume of the propositus measuring a whopping 17 fL. All affected individuals had heterozygous mutations: P53L in the gene encoding GPIbα (GP1BA), and D723H in the gene encoding integrin β3 (INTB3). At least 4 syndromes (Bernard-Soulier, DiGeorge, benign Mediterranean macrothrombocytopenia, and platelet-type von Willebrand disease) are associated with macrothrombocytopenia and GP1BA mutations. However, convincing data were provided to suggest that the GP1BA P53L mutation was not responsible for the phenotype in this pedigree, and molecular modeling predicted that the GP1BA P53L substitution would not affect von Willebrand factor binding. Platelets from subjects with the INTB3 D723H mutation showed spontaneous α_IIbβ₃ activation, and CHO cells expressing α_IIbβ₃-D723H displayed enhanced binding to immobilized fibrinogen. Intriguingly, in vitro–differentiated megakaryocytes with the β₃-D723H mutation exhibited larger proplatelet buds than the wild type.

It was not long ago that molecular genetic studies were rather primitive compared with the thorough multidisciplinary approaches used by Ghevaert and colleagues to characterize this pedigree. Several novel findings are presented and questions raised. First, integrin mutations should now be added to the list of causes of macrothrombocytopenia. Second, the naturally occurring mutation in β₃ supports the existence and function of the conserved intracellular salt bridge between α_IIb-R995 and β₃-D723 that was proposed using elegant in vitro charge-reversal mutation studies (see figure).³  Third, although the interaction of α_IIbβ₃ with fibrinogen stimulates proplatelet development,⁴  a mechanism that integrates the induction of proplatelet formation due to the β3-D723H mutation with the formation of large platelets remains to be established. Perhaps this phenotype results from a simple physical effect whereby increased proplatelet adhesion to bone marrow sinusoidal fibrinogen delays platelet release while size continues to increase. Or perhaps, since integrin binding to extracellular matrix induces gene transcription,⁵  the enhanced adhesion of β3-D723H induces expression of genes regulating platelet size or favors cross-talk with GPIbα, tubulin, or other cytoskeletal proteins.