Relationships between Rap1b, affinity modulation of integrin α_IIbβ₃ and the actin cytoskeleton

The affinity of integrin α_IIbβ₃for fibrinogen is controlled by inside-out signals that are triggered by agonists like thrombin. Agonist treatment of platelets also activates Rap1b, a small GTPase known to promote integrin-dependent adhesion of other cells. Therefore, we investigated the role of Rap1b in α_IIbβ₃function by viral transduction of GFP-Rapl chimeras into murine megakaryocytes, which exhibit inside-out signaling similar to platelets. Expression of constitutively active GFP-Rap1b (V12) had no effect on unstimulated megakaryocytes, but it greatly augmented fibrinogen binding to α_IIbβ₃induced by a PAR4 thrombin receptor agonist (p < 0.01). The Rap1b effect was cell-autonomous and was prevented by pre-treating cells with cytochalasin D or latrunculin A to inhibit actin polymerization. Raplb-dependent fibrinogen binding to megakaryocytes was blocked by POW-2, a novel monovalent antibody Fab fragment specific for high affinity murine α_IIbβ₃In contrast to GFP-Rap1b (V12), expression of GFP-RaplGAP, which deactivates endogenous Rapl, inhibited agonist-induced fibrinogen binding (p < 0.01), as did dominant-negative GFP-Rap1b (N17) (p < 0.05). None of these treatments affected surface expression of α_IIbβ₃. These studies establish that Rap1b can augment agonist-induced ligand binding to α_IIbβ₃ through effects on integrin affinity, possibly by modulating α_IIbβ₃interactions with the actin cytoskeleton.