OC 35.4 - Critical Role for RapGEF2 in PKC-Rap1-Integrin Signaling in Platelets

Background: The small GTPase Rap1 is a crucial regulator of platelet integrin activation, a critical event during the formation of both hemostatic and thrombotic plugs. Rap1 activation in response to cellular stimulation is controlled by guanine nucleotide exchange factors (GEFs). In platelets, CalDAG-GEFI (RasGRP2) is the most highly expressed and functionally dominant GEF. However, genome-wide association studies (GWAS) also suggest a significant role for RapGEF2 (PDZ-GEFI) in platelet integrin inside-out activation and coronary artery disease (CAD).

Aims: To examine the role of RapGEF2 in Rap1-integrin signaling in platelets.

Methods: In this study we used mice deficient in RapGEF2 (Rapgef2-mKO), CalDAG-GEFI (Caldaggef1-/-), or both (DKO) to characterize the contribution of both proteins to platelet function. In vitro assays of integrin activation, granule secretion, platelet aggregation and Rap1 activation were used to assess platelet activation, while in vivo platelet function was assessed by saphenous vein laser injury and ferric chloride carotid injury.

Results: Rapgef2-mKO platelets activated with various agonists showed minor defects in platelet integrin inside-out activation. In contrast, integrin-mediated aggregation in Caldaggef1-/- platelets occurred with a delay and was dependent on protein kinase C (PKC) signaling and RapGEF2. PKC/RapGEF2 signaling was found to be critical for sustained Rap1 signaling. Consistent with these in vitro findings, thrombosis and hemostatic plug formation were markedly impaired in Caldaggef1-/- but not Rapgef2-mKO mice. However, hemostatic plugs in DKO mice were more unstable and bleeding times were significantly longer when compared to Caldaggef1-/- mice.

Conclusion(s): In summary, here we provide genetic evidence that RapGEF2 operates downstream of PKC as an important regulator of sustained Rap1 signaling and integrin activation in platelets. Our findings are supportive of GWAS data suggesting a key role for RapGEF2 in platelet function and may have important implications for risk assessment and prevention strategies in CAD.