αIIb(R990W), a Constitutive Activating Mutation of Integrin αIIbβ3, Knock-in Mice Show Macrothrombocytopenia with Impairment of Platelet Function

Background Integrin αIIbβ3 plays an essential role in platelet aggregation and quantitative or qualitative defects in αIIbβ3 lead to severe bleeding diathesis, which is known as Glanzmann thrombasthenia (GT). Although platelet counts and morphology are usually normal in GT patients, we and others found that several mutations around transmembrane regions of αIIb or β3, which cause constitutive activation of αIIbβ3, are associated with macrothrombocytopenia. Among them, αIIb(R995W) was found in four unrelated Japanese families, suggesting that the mutation may be the most prevalent in Japanese regarding αIIbβ3-associated macrothrombocytopenia (Kunishima, Kashiwagi, et al. Blood. 2011;117:5479-84). We also found reduction of surface expression of αIIbβ3 on platelets in patients with αIIbβ3-associated macrothrombocytopenia, suggesting that activating mutations may affect platelet function via not only activating state of αIIbβ3 but also its expression (Kashiwagi, et al. Mol Genet Genomic Med. 2013;1:77-86). In this study, we generated αIIb(R990W), which is corresponding to human αIIb(R995W), knock-in (KI) mice and analyzed the effects of constitutive activation of αIIbβ3 on platelet numbers, morphology and function.

Methods: αIIb(R990W) mutation was introduced to C57BL/6-derived ES cells by homologous recombination and KI mice were generated. Reticulated platelets were detected by flow cytometry after thiazole orange staining. Platelet life span was assessed by flow cytometry after injection with NHS-biotin. Platelet function was assessed by light transmission aggregometry and platelet adhesion/aggregation on collagen-coated glass under shear stress in vitro. Platelet function was also assessed by tail bleeding time and thrombus formation induced by FeCl₃injury on mesenteric artery in vivo.

Results: Hetero and Homo KI mice were born in accordance with Mendel’s laws and showed no obvious bleeding tendency. Platelet counts were decreased in KI mice [WT: 1.22±0.11(x10⁶/μl), Hetero: 1.10±0.14*, Homo: 0.91±0.11* (*p<0.05 compared with WT)] and platelet size assessed by forward side scatter of flow cytometry showed mild increase of platelet size in KI mice [WT: 26.3±0.60 (arbitrary unit), Hetero: 27.7±0.56*, Homo: 32.5±0.98*(*p<0.05 compared with WT)]. Absolute reticulated platelet numbers of Homo were decreased compared with WT [WT: 102±21.9 (x10³/μl), Hetero: 84.0±23.7, Homo: 66.6±12.3*(*p<0.05 compared with WT)]. There was no difference in serum thrombopoietin levels among Homo, Hetero and WT mice. Platelet recovery after thrombocytopenia caused by anti-GPIb antibodies was mildly impaired on 4 days after injection in KI mice. These results suggest that platelet production may be mildly impaired in KI mice. Platelet life span was also slightly but significantly shortened in KI mice compared with WT, which may also contribute to thrombocytopenia in KI mice. Surface expression of αIIbβ3 in Hetero and Homo platelets was decreased to 74.0±3.4 and 3.3±0.6% of WT, whereas that on megakaryocytes in Hetero and Homo was 81.0±15.2% and 62.3±8.9% of WT, respectively. Bleeding time of Homo mice was markedly prolonged [WT: 2.44±2.08 min (n=12), Hetero: 3.14±2.62 min (n=12), Homo: >10min in 8 of 10 mice]. Thrombus formation on collagen-coated coverslip under flow conditions as well as platelet aggregation induced by collagen and PAR4-TRAP was markedly impaired in Homo platelets. Homo mice also showed markedly impaired thrombus formation in FeCl₃-induced thrombus model. There were no significant differences in platelet function between Hetero and WT.

Conclusion: The αIIbβ3 activating mutation, αIIb(R990W), leads to macrothrombocytopenia in mice. Homo KI mice also show marked reduction of surface αIIbβ3 expression in platelet, leading to severe impairment of platelet aggregation and thrombus formation like GT.