β3-Deficient megakaryocytes were modified by human β3-lentivirus transduction and transplantation to express sufficient levels of a C560Rβ3 amino acid substitution, for investigation of how an activated αIIbβ3 conformation affects plateletsin vivo in mice.
Patient/Methods
As in our previous report of an R560β3 mutation in a patient with Glanzmann thrombasthenia, R560β3 murine platelets spontaneously bound antibody that only recognizes activated αIIbβ3 bound to its ligand, fibrinogen.
Results
With this murine model, we showed that αIIb-R560β3 mutation-mediated continuous binding of fibrinogen occurred in the absence of P-selectin surface expression, indicating that the integrin was in an active conformation, although the platelets circulated in a quiescent manner. Remarkably, only 35% of R560β3 ‘mutant’ mice survived for 6 months after transplantation, whereas 87% of C560β3 ‘wild-type’ mice remained alive. Pathologic examination revealed that R560β3 mice had enlarged spleens with extramedullary hematopoiesis and increased hemosiderin, indicating hemorrhage. R560β3 megakaryocytes and platelets showed abnormal morphology and irregular granule distribution. Interestingly, R560β3 washed platelets could aggregate upon simultaneous addition of fibrinogen and physiologic agonists, but aggregation failed when platelets were exposed to fibrinogen before activation in vitro and in vivo.
Conclusions
The results demonstrate that continuous occupancy of αIIbβ3 with fibrinogen disrupts platelet structure and function, leading to hemorrhagic death consistent with Glanzmann thrombasthenia rather than a thrombotic state.
