CalDAG-GEFI Is a Key Signal Integrator in Platelet Activation and Thrombus Formation.

Inside-out activation of platelet integrin αIIbβ3 is a key step in agonist-induced platelet aggregation. Recent studies suggested the involvement of the small GTPase Rap1b in this process as it is highly expressed in platelets and becomes activated during platelet activation. In cell lines, overexpression of the Rap activator CalDAG-GEFI increased αIIbβ3-dependent adhesion, while overexpression of RapGAP, which inactivates Rap1, reduced αIIbβ3 activity. Here we provide evidence that CalDAG-GEFI is an essential component of this pathway in vivo. To generate CalDAG-GEFI knockout mice, we engineered mouse embryonic stem (ES) cells with a deletion that results in a frameshift mutation and a premature stop codon at the position encoding the 37th amino acid of CalDAG-GEFI. These ES cells were then used to derive chimeric mice that yielded germline transmission of the CalDAG-GEFI mutation. Deficiency of CalDAG-GEFI in mutant mice was confirmed by immunohistochemistry and western blot analysis. CalDAG-GEFI^−/− platelets showed impaired Rap1b activation and aggregation in response to various agonists, with aggregation being completely blocked when platelets were activated with ADP, thromboxaneA2 analog, or calcium ionophore. Under physiological flow conditions in vitro and in vivo, CalDAG-GEFI-deficient platelets showed normal tethering to basement membrane components but failed to form thrombi. Mice deficient in CalDAG-GEFI were further characterized by a greatly increased bleeding time as well as by a strong protection against collagen-induced pulmonary thrombosis. In summary, we identified CalDAG-GEFI as a key signal integrator in the cascade leading through Rap1 and integrin αIIbβ3 to platelet aggregation and thrombus formation. The fact that CalDAG-GEFI knockout mice are resistant to collagen-induced thrombosis, and do not undergo spontaneous hemorrhaging, suggests that CalDAG-GEFI may be a promising new target for antithrombotic therapy.