Identification of Integrin β3 L718P Mutation in a Pedigree with Autosomal Dominant Macrothrombocytopenia

Abstract 1081

α IIbβ3 integrin mutations that result in the complete loss of expression of this molecule on the platelet surface cause Glanzmann's thrombasthenia. This is usually an autosomal recessive, while other mutations are known to cause dominantly inherited macrothrombocytopenia (although such cases are rare). Here, we report a 4-generation pedigree including 10 individuals affected by dominantly inherited macrothrombocytopenia. Six individuals, whose detailed clinical and laboratory data are available, carry a non-synonymous ITGB3gene T2231C alteration resulting in the substitution of leucine at 718 for proline (L718P) in the integrin β3 protein.

The patient was 4-year old Japanese girl, who presented with mild bleeding tendency. Her platelet count was 49–72 x10⁹/L with a mean platelet volume of 6.7–10.4 fl. WBC and RBC numbers were normal and there were no morphological abnormalities including inclusions in neutrophils. In the patient, marked platelet anisocytosis and giant platelets were observed in peripheral blood samples. Platelet aggregation induced by ADP and collagen was markedly reduced, but agglutination induced by ristocetin was within the normal range. The α IIbβ3 expression level determined by flow cytometry in the patient was 50–60% of the healthy control. A total of six of her relatives were subsequently found to have low platelet counts and were referred to our institute for further investigation.

To isolate a candidate gene alteration responsible for the macrothrombocytopenia, whole exome sequencing analysis was performed using genomic DNA obtained from four affected individuals of the pedigree including the patient. Among the 90 non-synonymous alterations commonly found in the affected individuals, we focused on the heterozygous integrin β3-L718P mutation, because this was recently reported as a candidate mutation responsible for macrothrombocytopenia (Jayo et al, 2010). As far as we could determine, no other non-synonymous gene alterations previously reported to cause thrombocytopenia or defective platelet function were present in the affected individuals of the pedigree.

Resting platelets from affected individuals showed a mild but significant increase of a ligand-mimicking PAC-1 binding relative to healthy individuals. However, in ADP-treated platelets carrying the mutation, only a small increase of affinity to PAC-1 was observed. These findings suggest that α IIbβ3-L718P is partially activated in the absence of inside-out signals such as ADP, but nevertheless cannot be fully activated in the presence of stimulating signals.

As previously reported by others, CHO cells expressing α IIbβ3-L718P formed long proplatelet-like protrusions on fibrinogen-coated dishes. This was reported to be mediated by the downregulation of RhoA activity, which is initiated by the binding of c-Src to the C-terminal tail of integrin β3. Indeed, we found that the formation of long cell protrusion was inhibited, when a constitutively-active form of RhoA (Q63L) was introduced into α IIbβ3-L718P-expressing cells. In addition, CHO cells expressing α IIbβ3-L718P (del. 759) mutant, which lacks the C-terminal c-Src binding site of integrin β3, did not form any proplatelet-like protrusions. However, because the enforced expression of a dominant negative form of RhoA (T19N) in α IIbβ3-WT expressing cells did not show typical proplatelet-like protrusions, it is suggested that downregulation of RhoA was required but not sufficient for the formation of proplatelet-like protrusions induced by integrin β3-L718P.

In summary, identification of a pedigree showing autosomal dominant inheritance leads to a model whereby the integrin β3-L718P mutation contributes to macrothrombocytopenia most likely through gain-of-function mechanisms.