RESUMO
Regulation of the platelet actin cytoskeleton by the Rho family of small GTPases is essential for the proper maintenance of hemostasis. However, little is known about how intracellular platelet activation from Rho GTPase family members, including Rac, Cdc42, and Rho, translate into changes in platelet actin structures. To better understand how Rho family GTPases coordinate platelet activation, we identified platelet proteins associated with Rac1, a Rho GTPase family member, and actin regulatory protein essential for platelet hemostatic function. Mass spectrometry analysis revealed that upon platelet activation with thrombin, Rac1 associates with a set of effectors of the p21-activated kinases (PAKs), including GIT1, ßPIX, and guanine nucleotide exchange factor GEFH1. Platelet activation by thrombin triggered the PAK-dependent phosphorylation of GIT1, GEFH1, and other PAK effectors, including LIMK1 and Merlin. PAK was also required for the thrombin-mediated activation of the MEK/ERK pathway, Akt, calcium signaling, and phosphatidylserine (PS) exposure. Inhibition of PAK signaling prevented thrombin-induced platelet aggregation and blocked platelet focal adhesion and lamellipodia formation in response to thrombin. Together, these results demonstrate that the PAK signaling system is a key orchestrator of platelet actin dynamics, linking Rho GTPase activation downstream of thrombin stimulation to PAK effector function, MAP kinase activation, calcium signaling, and PS exposure in platelets.
Assuntos
Plaquetas/efeitos dos fármacos , Ativação Plaquetária/efeitos dos fármacos , Trombina/farmacologia , Quinases Ativadas por p21/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Plaquetas/citologia , Plaquetas/metabolismo , Adesão Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Regulação da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Quinases Lim/genética , Quinases Lim/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neurofibromina 2/genética , Neurofibromina 2/metabolismo , Fosforilação , Agregação Plaquetária/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pseudópodes/efeitos dos fármacos , Pseudópodes/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho , Transdução de Sinais , Quinases Ativadas por p21/genética , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
OBJECTIVE: Rho GTPase proteins play a central role in regulating the dynamics of the platelet actin cytoskeleton. Yet, little is known regarding how Rho GTPase activation coordinates platelet activation and function. In this study, we aimed to characterize the role of the Rho GTPase effector, p21 activated kinase (PAK), in platelet activation, lamellipodia formation, and aggregate formation under shear. APPROACH AND RESULTS: Stimulation of platelets with the glycoprotein receptor VI agonist, collagen-related peptide, rapidly activated PAK in a time course preceding phosphorylation of PAK substrates, LIM domain kinase LIMK1 and the MAPK/ERK kinase MEK, and the subsequent activation of MAPKs and Akt. Pharmacological inhibitors of PAK blocked signaling events downstream of PAK and prevented platelet secretion as well as platelet aggregation in response to collagen-related peptide. PAK inhibitors also prevented PAK activation and platelet spreading on collagen surfaces. PAK was also required for the formation of platelet aggregates and to maintain aggregate stability under physiological shear flow conditions. CONCLUSIONS: These results suggest that PAK serves as an orchestrator of platelet functional responses after activation downstream of the platelet collagen receptor, glycoprotein receptor VI.
Assuntos
Plaquetas/enzimologia , Ativação Plaquetária , Agregação Plaquetária , Glicoproteínas da Membrana de Plaquetas/metabolismo , Pseudópodes/enzimologia , Transdução de Sinais , Quinases Ativadas por p21/sangue , Plaquetas/efeitos dos fármacos , Proteínas de Transporte/farmacologia , Forma Celular , Ativação Enzimática , Humanos , Quinases Lim/sangue , MAP Quinase Quinase Quinases/sangue , Peptídeos/farmacologia , Fosforilação , Ativação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Glicoproteínas da Membrana de Plaquetas/agonistas , Inibidores de Proteínas Quinases/farmacologia , Pseudópodes/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Estresse Mecânico , Fatores de Tempo , Quinases Ativadas por p21/antagonistas & inibidoresRESUMO
The characterization of platelet aggregation and thrombus formation typically requires the use of fluorescent labels followed by fluorescent confocal microscopy. However, fluorescent labels have been suspected to affect platelet function. We have developed a label-free imaging technique to characterize the volume and surface area coverage of platelet aggregates and thrombi formed under shear. Platelet aggregates were formed by perfusing anti-coagulated whole blood over fibrillar collagen. Thrombi were formed by perfusing recalcified whole blood over fibrillar collagen in the presence of coagulation. Platelet aggregates and thrombi volume and surface area coverage were quantified using a Hilbert transform differential interference contrast (DIC) microscopy technique (HT-DIC). Our data indicate that platelet aggregates and thrombi formed at a shear rate of 200 s-1 had similar volume and surface area coverage. At a shear rate of 1000 s-1, both the volume and surface area coverage of platelet aggregates significantly increased as compared to low shear conditions. In contrast, the volume of thrombi formed in the presence of coagulation appeared to remain the same at both low and high shear rates. Utilization of this HT-DIC imaging technique can allow for insights into the kinetics and mechanisms by which thrombi are formed under various shear conditions in a label-free manner.
