Rap1b is required for normal platelet function and hemostasis in mice
Journal of Clinical Investigation
Rap1b , an abundant small GTPase in platelets, becomes rapidly activated upon stimulation with agonists. Though it has been implicated to act downstream from G protein-coupled receptors (GPCRs) and upstream of integrin α IIb β 3 , the precise role of Rap1b in platelet function has been elusive. Here we report the generation of a murine rap1b knockout and show that Rap1b deficiency results in a bleeding defect due to defective platelet function. Aggregation of Rap1b-null platelets is reduced in
... lets is reduced in response to stimulation with both GPCR-linked and GPCR-independent agonists. Underlying the defective Rap1b-null platelet function is decreased activation of integrin α IIb β 3 in response to stimulation with agonists and signaling downstream from the integrin α IIb β 3 . In vivo, Rap1b-null mice are protected from arterial thrombosis. These data provide genetic evidence that Rap1b is involved in a common pathway of integrin activation, is required for normal hemostasis in vivo, and may be a clinically relevant antithrombotic therapy target. Results Generation of Rap1b-deficient mice. Rap1b-null mice were created by replacing the entire coding sequence of the murine rap1b gene with a neomycin-resistance gene (Figure 1 ). Of the first 91 mice generated from the rap1b +/intercrosses, at weaning, 42.9% were rap1b +/+ , 52.7% were rap1b +/-, and 4.4% were rap1b -/-, indicating 85% embryonic and perinatal lethality of the Rap1b-null mice. Analysis of staged embryos derived from heterozygous intercrosses revealed that at least 40% of rap1b -/embryos had abdominal, cranial, and hepatic bleeding ( Figure 1F ) and died in utero between E13.5 and E18.5, while the remaining deaths occurred perinatally. Surviving Rap1b-null mice were smaller than their littermates but appeared otherwise normal, with no overt cancer or hematological defects and normal life spans. Rap1b-null mice were fertile and produced 45% smaller litters at weaning compared with wild-type mice Nonstandard abbreviations used: GPCR, G protein-coupled receptor; GPVI, glycoprotein GPVI; PKA, cAMP-dependent protein kinase; PLCβ, phospholipase Cβ; PRP, platelet-rich plasma; VASP, vasodilator-stimulated phosphoprotein.