A novel heterozygous ITGB3 p.T720del inducing spontaneous activation of integrin αIIbβ3 in autosomal dominant macrothrombocytopenia with aggregation dysfunction

Abstract

We identified a novel heterozygous ITGB3 p.T720del mutation in a pedigree with macrothrombocytopenia exhibiting aggregation dysfunction. Platelet aggregation induced by ADP and collagen was significantly reduced, while ristocetin aggregation was normal. Integrin αIIbβ3 was partially activated in a resting status, but platelet expression of αIIbβ3 was downregulated. Functional analysis using a cell line showed spontaneous phosphorylation of FAK in αIIb/β3 (p.T720del)-transfected 293T cells in suspension conditions. Abnormal cytoplasmic protrusions, membrane ruffling, and cytoplasmic localization of αIIbβ3 were observed in αIIb/β3 (p.T720del)-transfected CHO cells. Such morphological changes were reversed by treatment with an FAK inhibitor. These findings imply spontaneous, but partial, activation of αIIbβ3 followed by phosphorylation of FAK as the initial mechanism of abnormal thrombopoiesis. Internalization and decreased surface expression of αIIbβ3 would contribute to aggregation dysfunction. We reviewed the literature of congenital macrothrombocytopenia associated with heterozygous ITGA2B or ITGB3 mutations. Reported mutations were highly clustered at the membrane proximal region of αIIbβ3, which affected the critical interaction between αIIb R995 and β3 D723, resulting in a constitutionally active form of the αIIbβ3 complex. Macrothrombocytopenia caused by a heterozygous activating mutation of ITGA2B or ITGB3 at the membrane proximal region forms a distinct entity of rare congenital thrombocytopenia.

Knock‐in mice bearing constitutively active αIIb(R990W) mutation develop macrothrombocytopenia with severe platelet dysfunction

“…Mutations in this region lead to constitutive activation of αIIbβ3 and induce aberrant outside‐in signaling via αIIbβ3 . Recent genetic studies on congenital macrothrombocytopenia suggest that gain‐of‐function mutations of αIIbβ3 in this region may affect not only platelet function but also platelet counts and morphology, which is referred to as αIIbβ3 ( ITGA2B/ITGB3 )‐related macrothrombocytopenia . In the present study, we showed that the αIIb(R990W) mutation in mice corresponding to αIIb(R995W) in humans, which is the most prevalent mutation in αIIbβ3‐related thrombocytopenia in Japan, induces mild thrombocytopenia with enlarged platelet size and downregulation of αIIbβ3 expression in platelets, leading to GT‐like severe platelet dysfunction in homo mice.…”

“…Abnormal proplatelet formation was observed in constitutively active αIIbβ3‐transduced megakaryocytes, as well as megakaryocytes obtained from patients with αIIbβ3‐related macrothrombocytopenia . Although the precise mechanisms remain to be elucidated, previous studies suggested that constitutive activation of focal adhesion kinase induced by the active form of αIIbβ3 causes abnormal actin remodeling via inhibition of RhoA activation . Furthermore, our results showed that platelet lifespan was slightly but significantly shortened in homo mice (Figure C), which could in turn partially affect thrombocytopenia in KI mice.…”

“…However, we detected high surface and total αIIbβ3 expression levels in the megakaryocytes and proplatelets of homo mice (Figure B, C), which suggested that mechanisms other than αIIbβ3 biosynthesis could be responsible for the observed reduction in αIIbβ3 levels in the platelets of KI mice. Increased internalization and degradation of αIIbβ3 was demonstrated with other gain‐of‐function mutations of αIIbβ3; therefore, we examined αIIbβ3 expression in platelets after anti‐CD42b MoAb injection because these samples are expected to contain a high percentage of newly synthesized young platelets. αIIbβ3 expression in homo platelets was increased to 10% to 15% of WT platelets on day 4 after anti‐CD42b MoAb injection from ~3% of WT in steady states (Figure S3 in supporting information).…”

“…Although platelet counts and morphology are usually normal in GT patients and αIIbβ3‐deficient mice or dogs, several αIIb or β3 mutations have been recently identified in patients with congenital macrothrombocytopenia, which is referred to as αIIbβ3( ITGA2B/ITGB3 )‐related macrothrombocytopenia. The above findings suggested that in addition to platelet function, αIIbβ3 is involved in platelet production and morphology . Interestingly, mutations in αIIbβ3 lead to a constitutively active conformation with aberrant outside‐in signaling via αIIbβ3 .…”

“…The above findings suggested that in addition to platelet function, αIIbβ3 is involved in platelet production and morphology . Interestingly, mutations in αIIbβ3 lead to a constitutively active conformation with aberrant outside‐in signaling via αIIbβ3 . Among these mutations, the αIIb(R995W) mutation, which is located just beneath the transmembrane domain of αIIb and induces the active conformation of αIIbβ3 by perturbing a salt bridge between αIIb(R995) and β3(D723), has been identified in at least four unrelated Japanese families with macrothrombocytopenia.…”