Calcium-diacylglycerol guanine nucleotide exchange factor I gene mutations associated with loss of function in canine platelets

Abstract

Calcium-Diacylglycerol Guanine Nucleotide Exchange Factor I (CalDAG-GEFI) has been implicated in platelet aggregation signaling in CalDAG-GEFI knockouts. Functional mutations were identified in the gene encoding for CalDAG-GEFI in 3 dog breeds. Affected dogs experienced epistaxis, gingival bleeding, and petechiation. Platelet number, von Willebrand factor, clot retraction, and coagulation screening assays were normal, whereas bleeding time tests were prolonged. Platelet aggregation and release responses to all agonists, except thrombin, were markedly impaired. Platelet membranes had normal concentrations of integrin alphaIIb-beta3; however, ADP-induced fibrinogen binding by activated platelets was markedly impaired. Forskolin-stimulated platelets exhibited a marked increase in intraplatelet cAMP associated with impaired phosphodiesterase (PDE) activity, whereas levels of extractable phosphoinositides were 1.5-fold to 2-fold higher in thrombin-stimulated affected platelets. DNA analysis of the CalDAG-GEFI gene in affected dogs documented the existence of 3 distinct mutations within portions of the CalDAG-GEFI gene encoding for structurally conserved regions within the catalytic domain of the protein. The mutations are predicted to result in either lack of synthesis, enhanced degradation, or marked impairment of protein function. The dysfunctional profile of canine platelets observed in mutant dogs putatively links CalDAG-GEFI and its target Rap1 or other Ras family member, for the first time, to a role in pathways that regulate cAMP PDE activity and thrombin-stimulated phosphoinositide anchoring or metabolism. The finding of distinct functional mutations in 3 dog breeds suggests that mutations in the CalDAG-GEFI gene may be implicated in similar defects in human patients with congenital platelet disorders having primary secretion defects of unknown etiology.