Jiaqing Pang

S6K1 and mTOR regulate Rac1-driven platelet activation and aggregation

Platelet activation and thrombus formation are under the control of signaling systems that integrate cellular homeostasis with cytoskeletal dynamics. Here, we identify a role for the ribosome protein S6 kinase (S6K1) and its upstream regulator mTOR in the control of platelet activation and aggregate formation under shear flow. Platelet engagement of fibrinogen initiated a signaling cascade that triggered the activation of S6K1 and Rac1. Fibrinogen-induced S6K1 activation was abolished by inhibitors of Src kinases, but not Rac1 inhibitors, demonstrating that S6K1 acts upstream of Rac1. S6K1 and Rac1 interacted in a protein complex with the Rac1 GEF TIAM1 and colocalized with actin at the platelet lamellipodial edge, suggesting that S6K1 and Rac1 work together to drive platelet spreading. Pharmacologic inhibitors of mTOR and S6K1 blocked Rac1 activation and prevented platelet spreading on fibrinogen, but had no effect on Src or FAK kinase activation. mTOR inhibitors dramatically reduced collagen-induced platelet aggregation and promoted the destabilization of platelet aggregates formed under shear flow conditions. Together, these results reveal novel roles for S6K1 and mTOR in the regulation of Rac1 activity and provide insights into the relationship between the pharmacology of the mTOR system and the molecular mechanisms of platelet activation.

Histone deacetylase 6-mediated deacetylation of α-tubulin coordinates cytoskeletal and signaling events during platelet activation

The tubulin cytoskeleton plays a key role in maintaining the characteristic quiescent discoid shape of resting platelets. Upon activation, platelets undergo a dramatic change in shape; however, little is known of how the microtubule system contributes to regulating platelet shape and function. Here we investigated the role of the covalent modification of α-tubulin by acetylation in the regulation of platelet physiology during activation. Superresolution microscopy analysis of the platelet tubulin cytoskeleton showed that the marginal band together with an interconnected web of finer tubulin structures collapsed upon platelet activation with the glycoprotein VI (GPVI)-agonist collagen-related peptide (CRP). Western blot analysis revealed that α-tubulin was acetylated in resting platelets and deacetylated during platelet activation. Tubacin, a specific inhibitor of the tubulin deacetylase HDAC6, prevented tubulin deacetylation upon platelet activation with CRP. Inhibition of HDAC6 upregulated tubulin acetylation and disrupted the organization of the platelet microtubule marginal band without significantly affecting platelet volume changes in response to CRP stimulation. HDAC6 inhibitors also inhibited platelet aggregation in response to CRP and blocked platelet signaling events upstream of platelet Rho GTPase activation. Together, these findings support a role for acetylation signaling in controlling the resting structure of the platelet tubulin marginal band as well as in the coordination of signaling systems that drive platelet cytoskeletal changes and aggregation.

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

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.

p21 Activated Kinase Signaling Coordinates Glycoprotein Receptor VI–Mediated Platelet Aggregation, Lamellipodia Formation, and Aggregate Stability Under Shear

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.

Oral administration of Bruton's tyrosine kinase inhibitors impairs GPVI-mediated platelet function

The Tec family kinase Brutons tyrosine kinase (Btk) plays an important signaling role downstream of immunoreceptor tyrosine-based activation motifs in hematopoietic cells. Mutations in Btk are involved in impaired B-cell maturation in X-linked agammaglobulinemia, and Btk has been investigated for its role in platelet activation via activation of the effector protein phospholipase Cγ2 downstream of the platelet membrane glycoprotein VI (GPVI). Because of its role in hematopoietic cell signaling, Btk has become a target in the treatment of chronic lymphocytic leukemia and mantle cell lymphoma; the covalent Btk inhibitor ibrutinib was recently approved by the US Food and Drug Administration for treatment of these conditions. Antihemostatic events have been reported in some patients taking ibrutinib, although the mechanism of these events remains unknown. We sought to determine the effects of Btk inhibition on platelet function in a series of in vitro studies of platelet activation, spreading, and aggregation. Our results show that irreversible inhibition of Btk with two ibrutinib analogs in vitro decreased human platelet activation, phosphorylation of Btk, P-selectin exposure, spreading on fibrinogen, and aggregation under shear flow conditions. Short-term studies of ibrutinib analogs administered in vivo also showed abrogation of platelet aggregation in vitro, but without measurable effects on plasma clotting times or on bleeding in vivo. Taken together, our results suggest that inhibition of Btk significantly decreased GPVI-mediated platelet activation, spreading, and aggregation in vitro; however, prolonged bleeding was not observed in a model of bleeding.

Aspirin therapy reduces the ability of platelets to promote colon and pancreatic cancer cell proliferation: implications for the oncoprotein c-MYC.

Aspirin, an anti-inflammatory and antithrombotic drug, has become the focus of intense research as a potential anticancer agent owing to its ability to reduce tumor proliferation in vitro and to prevent tumorigenesis in patients. Studies have found an anticancer effect of aspirin when used in low, antiplatelet doses. However, the mechanisms through which low-dose aspirin works are poorly understood. In this study, we aimed to determine the effect of aspirin on the cross talk between platelets and cancer cells. For our study, we used two colon cancer cell lines isolated from the same donor but characterized by different metastatic potential, SW480 (nonmetastatic) and SW620 (metastatic) cancer cells, and a pancreatic cancer cell line, PANC-1 (nonmetastatic). We found that SW480 and PANC-1 cancer cell proliferation was potentiated by human platelets in a manner dependent on the upregulation and activation of the oncoprotein c-MYC. The ability of platelets to upregulate c-MYC and cancer cell proliferation was reversed by an antiplatelet concentration of aspirin. In conclusion, we show for the first time that inhibition of platelets by aspirin can affect their ability to induce cancer cell proliferation through the modulation of the c-MYC oncoprotein.

Characterization of the Rac guanine nucleotide exchange factor P-Rex1 in platelets

Background Blood platelets undergo a carefully regulated change in shape to serve as the primary mediators of hemostasis and thrombosis. These processes manifest through platelet spreading and aggregation and are dependent on platelet actin cytoskeletal changes orchestrated by the Rho GTPase family member Rac1. To elucidate how Rac1 is regulated in platelets, we captured Rac1-interacting proteins from platelets and identified Rac1-associated proteins by mass spectrometry. Findings Here, we demonstrate that Rac1 captures the Rac guanine nucleotide exchange factor P-Rex1 from platelet lysates. Western blotting experiments confirmed that P-Rex1 is expressed in platelets and associated with Rac1. To investigate the functional role of platelet P-Rex1, platelets from P-Rex1-/--deficient mice were treated with platelet agonists or exposed to platelet activating surfaces of fibrinogen, collagen and thrombin. Platelets from P-Rex1-/- mice responded to platelet agonists and activating surfaces similarly to wild type platelets. Conclusions These findings suggest that P-Rex1 is not required for Rac1-mediated platelet activation and that the GEF activities of P-Rex1 may be more specific to GPCR chemokine receptor mediated processes in immune cells and tumor cells.

Lysine acetyltransfer supports platelet function.

The reversible acetylation of protein lysine ε‐amino groups, catalyzed by lysine acetyltransferases and deacetylases, serves as a molecular switch in the orchestration of diverse cellular activities. Here, we aimed to investigate the role of lysine acetyltransfer in platelet function.

Assessment of roles for the Rho-specific guanine nucleotide dissociation inhibitor Ly-GDI in platelet function: a spatial systems approach

On activation at sites of vascular injury, platelets undergo morphological alterations essential to hemostasis via cytoskeletal reorganizations driven by the Rho GTPases Rac1, Cdc42, and RhoA. Here we investigate roles for Rho-specific guanine nucleotide dissociation inhibitor proteins (RhoGDIs) in platelet function. We find that platelets express two RhoGDI family members, RhoGDI and Ly-GDI. Whereas RhoGDI localizes throughout platelets in a granule-like manner, Ly-GDI shows an asymmetric, polarized localization that largely overlaps with Rac1 and Cdc42 as well as microtubules and protein kinase C (PKC) in platelets adherent to fibrinogen. Antibody interference and platelet spreading experiments suggest a specific role for Ly-GDI in platelet function. Intracellular signaling studies based on interactome and pathways analyses also support a regulatory role for Ly-GDI, which is phosphorylated at PKC substrate motifs in a PKC-dependent manner in response to the platelet collagen receptor glycoprotein (GP) VI-specific agonist collagen-related peptide. Additionally, PKC inhibition diffuses the polarized organization of Ly-GDI in spread platelets relative to its colocalization with Rac1 and Cdc42. Together, our results suggest a role for Ly-GDI in the localized regulation of Rho GTPases in platelets and hypothesize a link between the PKC and Rho GTPase signaling systems in platelet function.

Potentiation of TRAP-6-induced platelet dense granule release by blockade of P2Y12 signaling with MRS2395

Abstract The release of ADP from platelet dense granules and its binding to platelet P2Y12 receptors is key to amplifying the initial hemostatic response and propagating thrombus formation. P2Y12 has thus emerged as a therapeutic target to safely and effectively prevent secondary thrombotic events in patients with acute coronary syndrome or a history of myocardial infarction. Pharmacological inhibition of P2Y12 receptors represents a useful approach to better understand the signaling mediated by these receptors and to elucidate the role of these receptors in a multitude of platelet hemostatic and thrombotic responses. The present work examined and compared the effects of four different P2Y12 inhibitors (MRS2395, ticagrelor, PSB 0739, and AR-C 66096) on platelet function in a series of in vitro studies of platelet dense granule secretion and trafficking, calcium generation, and protein phosphorylation. Our results show that in platelets activated with the PAR-1 agonist TRAP-6 (thrombin receptor-activating peptide), inhibition of P2Y12 with the antagonist MRS2395, but not ticagrelor, PSB 0739 or AR-C 66096, potentiated human platelet dense granule trafficking to the plasma membrane and release into the extracellular space, cytosolic Ca2+ influx, and phosphorylation of GSK3β-Ser9 through a PKC-dependent pathway. These results suggest that inhibition of P2Y12 with MRS2395 may act in concert with PAR-1 signaling and result in the aberrant release of ADP by platelet dense granules, thus reducing or counteracting the anticipated anti-platelet efficacy of this inhibitor.