The PAK system links Rho GTPase signaling to thrombin-mediated platelet activation

Abstract

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, βPIX, 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.

Keywords: PAK; Rac1; Rho GTPases; actin; platelets.

Fig. 1.

Association of the Rac and p21-activated kinases (PAK) signaling systems in platelets. A: silver-stained polyacrylamide gel of Rac-associated proteins from thrombin-activated platelets identified by mass spectrometry (see Table 1). B: Western blot analysis of Rac1 captured GIT, PIX, and guanine nucleotide exchange factor GEFH1 proteins from thrombin-activated platelet lysates.

Fig. 2.

PAK activation in platelets upon stimulation with thrombin. A: human platelets treated with vehicle (DMSO), the Rho kinase (ROCK) inhibitor Y-27632 (10 μM), or the Rac inhibitor EHT 1864 (50 μM) before stimulation with 1 U/ml thrombin (Thr). Platelet PAK activation was determined by Western blot for phosphorylation of PAK2-Ser192 and phosphorylation of the PAK substrate GIT1-Ser517 under basal (lane 1) and thrombin-stimulated conditions (lanes 2–4). B: human platelets treated with vehicle (0.1%, DMSO), the inhibitor of PAK activation IPA-3 (10 μM), or the inactive PAK inhibitor relative PIR 3.5 (10 μM) before stimulation with 1 U/ml thrombin (Thr). PAK activation determined by Western blot for PAK2-pSer20, PAK2-pSer192 and PAK2-pThr402 under basal (lane 1) and thrombin-stimulated conditions (lanes 2-4). C: resting and thrombin-stimulated platelets treated with vehicle (0.1% DMSO), IPA-3 (10 μM), or PIR 3.5 (10 μM) were fixed in paraformaldehyde and examined for filopodia formation by differential interference contrast (DIC) microscopy. Scale bar = 1 μm. D: distribution analysis of measured platelet filopodia lengths from resting and thrombin-stimulated platelets treated with vehicle, IPA-3, or PIR 3.5 (n = 600 per condition).

Fig. 3.

Thrombin-elicited PAK, MEK/ERK, and Akt signaling requires PAK. Purified human platelets were treated with vehicle (0.1% DMSO), 10 μM IPA-3, or 10 μM PIR 3.5 for 10 min before stimulation with thrombin (1 U/ml, 5 min). Western blot analysis of PAK-dependent phosphorylation of Rac-captured PAK effectors (A) and classical PAK effectors (B) as well as MEK and ERK (C) and Akt (D). Western blot results are representative of 4 separate experiments.

Fig. 4.

Inhibition of PAK blocks platelet aggregation by thrombin. Replicate samples of washed human platelets (2 × 10⁸/ml) were incubated with vehicle (DMSO) or increasing concentrations of IPA-3 or 10 μM PIR 3.5 before stimulation with thrombin (0.07 U/ml), and the change in optical density indicative of platelet aggregation was recorded. Results are representative of 4 separate experiments.

Fig. 5.

PAK activation is required for platelet lamellipodia and actin-rich adhesion formation in response to thrombin. A: purified human platelets spread on a surface of thrombin, stained for PAK, Rac, and actin and visualized by fluorescence deconvolution microscopy. Scale bar = 2 μm. B: representative DIC images of human platelets treated with vehicle (DMSO), increasing concentrations of IPA-3 or 10 μM PIR 3.5 on a surface of thrombin. Scale bar = 10 μm. C: super-resolution analysis of platelet focal adhesion formation of vehicle and 10 μM IPA-3 treated platelets on a surface of thrombin, visualized by SR-SIM of actin and vinculin staining. Scale bar = 2 μm. D: purified human platelets were spread on a surface of thrombin. After 30 min, unbound platelets were removed and activated platelets were treated with vehicle (0.1% DMSO), 10 μM IPA-3, or 10 μM PIR 3.5 for an additional 15 min. Platelet spreading and lamellipodial withdrawal were evaluated by DIC microscopy. Scale bar = 10 μm.

Fig. 6.

PAK is required for platelet calcium signaling. A: platelets treated with vehicle, 10 μM IPA-3 or 10 μM PIR 3.5 on a surface of fibrinogen in the absence or presence of thrombin (1 U/ml) in solution. B: replicate samples of platelets on a surface of fibrinogen, loaded with Oregon Green BAPTA1-AM analyzed for calcium spiking in the absence or presence of thrombin (1 U/ml) in solution. C and D: visualization and quantification of PS exposure of platelets pretreated with vehicle, 10 μM IPA, or 10 μM IPR 3.5 on a surface of fibrinogen with thrombin in solution visualized by annexin V-FITC staining. Results are representative of 4 separate experiments.

Fig. 7.

A PAK-centric model of platelet activation. PAK links Rho GTPase family signaling to MAP kinase, Akt, and calcium systems to drive platelet lamellipodia formation and aggregation.