RAP1-RHO small GTPase cross-talk mediates integrindependent and -independent platelet procoagulant response.

Platelet adhesion and procoagulant activity are critical for primary and secondary hemostasis, respectively. The small GTPase RAP1 is a central regulator of platelet aggregation as it controls αIIbβ3 integrin activation through direct interaction with the integrin adapter protein, TALIN1 (TLN1). In addition to their aggregation defect, activated platelets lacking RAP1 (Rap1mKO) exhibited a marked impairment in surface exposure of phosphatidylserine (PtdSer), a negatively charged phospholipid with procoagulant activity. However, the mechanisms by which RAP1 regulates PtdSer exposure are unclear. Here we investigated the hypothesis that RAP1 regulates platelet PtdSer exposure through cross-talk with small GTPases of the RHO family. Consistent with their defect in PtdSer exposure, Rap1mKO platelets showed reduced procoagulant activity in vitro and in vivo when compared to controls. Stimulated Rap1mKO platelets exhibited elevated RHOA-GTP levels, and inhibition of the RHOA effector, Rho associated coiled-coil kinase (ROCK), partially restored PtdSer exposure in these cells. A milder defect in PtdSer exposure was observed for platelets from Tln1mR35/118E mice, i.e., mice with impaired RAP1-TLN1 interaction but otherwise intact RAP1 signaling. ROCK inhibition fully restored PtdSer exposure in Tln1mR35/118E platelets. Opening of the mitochondrial permeability transition pore, a cellular response critical to PtdSer exposure, was impaired in Rap- 1mKO platelets and restored by pretreatment of cells with the ROCK inhibitor. Our study provides first evidence that platelet RAP1 signaling affects hemostatic plug formation independent of its key role in platelet adhesion. Additionally, our studies strongly suggest that RAP1 regulates PtdSer exposure and procoagulant activity in a RHOA/integrin-dependent and -independent manner.