High Mortality in Mice with Platelets Expressing Integrin αIIbβ3 Lockedin Its High Affinity State.
Authors: Wilcox David A. , Fang Juan , North Paula , Nurden Paquita , Nurden Alan T. , Valentin Nathalie 
Published: April 2020

Abstract
A murine model was developed to investigate the effect of altered integrin β3 structure on αIIbβ3 activation and platelet function. To accomplish this, a lentivirus genetic transfer vector was adapted with the human αIIb megakaryocyte-specific gene promoter and cDNA encoding normal human β3 or an abnormal form with a substitution of C to R at amino acid 560. We previously characterized αIIbR560β3 as locked-in a high affinity state for ligand on the surface of platelets from a patient affected with a mild form of the inherited bleeding disorder, Glanzmann thrombasthenia (GT). Bone marrow was isolated from β3-deficient (−/−) mice, transduced with virions encoding either normal or the R560 form of human β3, and then transplanted into lethally irradiated β3(−/−) littermates. Following bone marrow transplant, flow cytometric analysis using antibodies to murine αIIb and human β3 detected expression of the hybrid murine αIIb-human β3 complex at similar levels on platelets from mice expressing either the normal or R560 form of human β3, although R560β3 platelets appeared slightly larger in size. A monoclonal antibody (D3) specific for the high affinity conformation of αIIbβ3 reacted readily with platelets expressing R560β3 in the absence of a fibrinogen mimetic peptide “RGD”, whereas platelets with normal β3 required pre-incubation with RGD for recognition by D3 indicating that αIIbR560β3 was locked-in a high affinity conformation on murine platelets. Murine platelets expressing normal β3 aggregated in response to treatment with adenosine diphosphate (ADP) alone or in a cocktail of agonists for platelet activation including ADP, epinephrine and thrombin receptor activating peptide. In contrast, R560β3 platelets only aggregated when treated with the cocktail of agonists, which is identical to aggregation studies with human platelets expressing R560β3. Interestingly, 50% of mice (n= 20) expressing R560β3 died between 1.5 and 11 weeks after bone marrow transplant while 95% of mice (n=18) expressing normal β3 survived up to that time point. Death was preceded by a rapid change in color of the extremities (including tail, paws and ears) to a pale white appearance and the animals became lethargic. Circulating blood counts showed that several animals had severe anemia upon death. Examination of organs (including brain, heart, kidney, lungs, liver, spleen and bowel) revealed that several of the deceased animals had enlarged spleens that were partially discolored, and some mice additionally had dark, necrotic-looking bowels. Histological examination of H&E stained spleen tissue revealed that the R560β3 mice had an increased level of hemosiderin and activated macrophages indicating that haemorrhage had occurred. This outcome suggests that locking integrin αIIbβ3 into its high affinity state may cause abnormal platelet thrombosis leading to haemorrhage, necrosis, severe anemia and rapid death of a significant number of mice (P=0.0025). A more complete analysis of the 40% of R560β3 mice that were able to survive for 0.5 years after transplant should help us to better understand why a GT patient survived for more than 45 years with the R560β3 gain-of-function mutation. Further investigations are underway to examine if the active structure of integrin αIIbR560β3 affects megakaryocytopoiesis and platelet formation as well as the platelet’s ability to bind to its major ligands. This work demonstrates the feasibility of using hematopoietic stem cell gene transfer to characterize altered forms of the human integrin αIIbβ3 within murine models.