PMC

As platelets land upon and attach to surfaces of adhesive proteins, such as fibrinogen, they spread upon surfaces by first forming actin-rich, finger-like filopodia (examples indicated with white arrow) which are then filled in by actin-rich sheets of lamellipodia (example indicated by white arc). Rho GTPase proteins RhoA, Cdc42 and Rac1 have specific roles in these actin-driven processes. Visualized by DIC microscopy []. Times shown in [min:sec]. Scale bar = 5 μm.

In their GTP-bound states, the Rho GTPases, including RhoA, Cdc42 and Rac1, associate with specific downstream effectors to regulate cytoskeletal remodeling events and platelet function. RhoA, Cdc42 and Rac1 are cyclically regulated by specific GEF and GAP proteins. GEFs promote GDP to GTP exchange to activate Rho GTPases. GAP proteins accelerate the hydrolysis of Rho-GTP to GDP and effectively inhibit Rho GTPase activation. Rho proteins may also be regulated by RhoGDI proteins, which “grab” and “release” Rho GTPases to sequester their activities [].

RhoA activation and inactivation drives separate steps of platelet activation. (A) Upon activation with thrombin, Gα_q activates p115RhoGEF to promote RhoA-GTP formation, ROCK activation and MLC phosphorylation to drive platelet shape change and secretion events. (B) Gα_q later supports the activation of c-Src through an association with the cytosolic domain of integrin α_IIbβ₃. c-Src activates p190RhoGAP to stimulate RhoA-GTP to GDP hydrolysis to effectively shut down RhoA contractile activity and facilitate platelet spreading. (C) Later, calcium signaling activates the calpain protease which cleaves integrin β₃, resulting in the loss of c-Src activity, driving platelet contraction and clot retraction [, , , ].

Upon stimulation of platelet GPCRs such as the PARs, TPs or P2Y12, Gα_q mediates the activation of Rac1 through PLCβ []. Integrin- and GPVI-coupled Src family kinase (SFK) activation also supports Rac1 activation [, , ]. GPIb release of 14-3-3ζ also has a role in platelet Rac1 activation []. G_i coupled to P2Y1 receptors is also required for full platelet Rac1 activation []. Separate pools of Rac1 may work in distinct steps of the platelet activation process. A first pool proximal to SFK-coupled receptor activation drives PLCγ activation and calcium signaling to activate CalDEG-GEF1 and the Rap1 GTPase to support integrin α_IIbβ₃ activation [, ]. A second pool of Rac1 downstream of PLCγ and calcium signaling then supports secretion, aggregation and spreading events through the activation of Rac effectors, such as the WAVE and Arp2/3 system or the PAK kinases.