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 collageninduced 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. (Blood. 2011;118(11):3129-3136) IntroductionPlatelets represent a specialized set of peripheral blood cells that are optimally configured for adhesion, secretion and aggregation at sites of vascular injury. 1,2 The exposure of platelets to extracellular matrix proteins such as collagen or laminin, or endogenous agonists such as ADP or thromboxanes, mediates hemostasis by activating signaling pathways that ultimately result in platelet adhesion and aggregation. 3 On the engagement of the adhesive proteins fibrinogen and fibronectin, platelet tyrosine kinases such as Src, Syk and FAK are recruited to the platelet cytosolic cell surface to initiate signaling pathways to drive platelet cytoskeletal reorganization through the Rho family small GTPase Rac1. [3][4][5] Rac1 regulates actin polymerization at the cell membrane to drive the growth and extension of platelet lamellipodiae that form the basis for platelet spreading. 4 The molecular mechanisms by which tyrosine kinases ultimately activate Rac1 remain ill-defined.The 70 kDa ribosome S6 protein kinase (S6K1) regulates the ribosome S6 protein to integrate processes of protein translation with cell growth and cell proliferation. 6 In cultured cells as well as in vivo, mitogenic signals triggered by nutrients and growth factors initiate a complex sequence of signaling events to activate the mammalian target of rapamycin (mTOR), a serine/threonine kinase which regulates S6K1 phosphorylation and activation. 7 Treatment of cells with rapamycin (Sirolimus) or other inhibitors of mTOR blocks S6K1 Thr389 phosphorylation and inhibits S6K1 activation. 8 The ability of mTOR inhibitors to arrest the growth of transformed tumor cells with...

The Tec family kinase Bruton's 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.

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.

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 GPVI agonist, collagen-related peptide (CRP), rapidly activated PAK in a time course preceding phosphorylation of PAK substrates LIMK1 and 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 CRP. 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 an orchestrator of platelet functional responses following activation downstream of the platelet collagen receptor, GPVI.

Background Treatment of chronic myelogenous leukemia (CML) with the BCR-ABL tyrosine kinase inhibitor (TKI) imatinib significantly improves patient outcomes. As some patients are unresponsive to imatinib, next generation BCR-ABL inhibitors such as nilotinib have been developed to treat patients with imatinib-resistant CML. The use of some BCR-ABL inhibitors has been associated with bleeding diathesis, and these inhibitors have been shown to inhibit platelet functions, which may explain the hemostasis impairment. Surprisingly, a new TKI, ponatinib, has been associated with a high incidence of severe acute ischemic cardiovascular events. The mechanism of this unexpected adverse effect remains undefined. Objective and Methods This study used biochemical and functional assays to evaluate whether ponatinib was different from the other BCR-ABL inhibitors with respect to platelet activation, spreading, and aggregation. Results and Conclusions Our results show that ponatinib, similar to other TKIs, acts as a platelet antagonist. Ponatinib inhibited platelet activation, spreading, granule secretion, and aggregation, likely through broad spectrum inhibition of platelet tyrosine kinase signaling, and also inhibited platelet aggregate formation in whole blood under shear. As our results indicate that pobatinib inhibits platelet function, the adverse cardiovascular events observed in patients taking ponatinib may be the result of the effect of ponatinib on other organs or cell types or disease-specific processes, such as BCR-ABL+ cells undergoing apoptosis in response to chemotherapy, or drug-induced adverse effects on the integrity of the vascular endothelium in ponatinib-treated patients.

Summary Background and Objectives 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. Methods and Results Proteomics methods identified 552 acetyllysine (acK) modifications on 273 platelet proteins that serve as candidate substrates for lysine acetyltransferases. Bioinformatics analyses of identified acK-modified platelet proteins supported roles for the lysine acetyltransferase p300 in the regulation of actin-mediated platelet processes. Biochemical experiments found that platelets express p300, which is activated in a Src kinase-dependent manner upon platelet stimulation with the platelet glycoprotein VI (GPVI) agonist CRP. Inhibition of platelet p300 abrogated CRP-stimulated lysine acetylation of actin, filamin and cortactin as well as F-actin polymerization, integrin activation and platelet aggregation. Super resolution visualization of platelet actin-rich adhesion structures revealed abundant acetyllysine protein content colocalized with platelet actin cytoskeletal proteins. Inhibition of p300 blocked platelet filopodia formation and the spreading of platelets on fibrinogen and collagen surfaces. In whole blood, p300 inhibition prevented the formation of platelet aggregates under shear, suggesting a physiological role for lysine acetyltransferase activity in platelet function. Conclusion Together, our findings uncover lysine acetyltransfer as a potential regulator of platelet actin dynamics and reveal potential roles for lysine acetylation in the molecular coordination of platelet activation and function.

BackgroundBlood 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.FindingsHere, 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.ConclusionsThese 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.

5261 An increase in mean platelet volume (MPV) is correlated with platelet activation and subsequent shape change. Pathologic processes marked by increased platelet activity such as myocardial infarction, cerebral vascular accidents, diabetes mellitus, and hypertension are associated with an increased MPV. Elevated MPV in these conditions reflect both a higher level of platelet activation as well as increased platelet turnover secondary to platelet consumption within thrombus formation. Assessment of MPV can be used to risk stratify patients as well as assign them to prognostic categories. However, MPV does not assess platelet heterogeneity or the specific change in single platelet mass, volume, or density. Current methods provide little insight into changes in physical parameters at the single platelet level. In order to overcome this limitation, we developed a quantitative tomographic differential interference contrast (QTDIC) microscopy technique to measure dry mass, volume, and density of platelets at the single-cell level. This technique is based on determining the axially resolved refractive index from a series of through-focus DIC images. Single cell platelet mass was observed to reduce from 1.84 ± 0.14 pg to 1.60 ± 0.13 pg in response to stimulation with thrombin-receptor agonist peptide (TRAP), while single cell platelet volume reduced from 7.28 ± 0.56 fL to 6.03 ± 0.48 fL (mean ± SEM). Single cell platelet density increased from 0.25 ± 0.001 pg/fL to 0.26 ± 0.002 pg/fL (mean ± SEM). Taken together, we have characterized the physical parameters of platelets in response to agonist stimulation. Our data suggest that platelet activation may correlate with decreased mass and volume, perhaps as a consequence of platelet degranulation. Further elucidation of the morphological changes of activated platelets at the single platelet level may allow for better understanding of platelet function and dysfunction in patients affected by platelet granule deficiencies, giant platelet syndromes, and disorders associated with membrane receptorsFigure 1.Characterization of physical parameters of platelets. (a) DIC image of human platelets, (b) refractive index map, (c) dry mass density map determined from refractive index using the Barer calibration, (d) Histogram of platelet dry mass, (e) Histogram of platelet volume, (f) Histogram of platelet density.Figure 1. Characterization of physical parameters of platelets. (a) DIC image of human platelets, (b) refractive index map, (c) dry mass density map determined from refractive index using the Barer calibration, (d) Histogram of platelet dry mass, (e) Histogram of platelet volume, (f) Histogram of platelet density. Disclosures: No relevant conflicts of interest to declare.