LDL oxidized by hypochlorous acid causes irreversible platelet aggregation when combined with low levels of ADP, thrombin, epinephrine, or macrophage-derived chemokine (CCL22)

Blood ◽  
2004 ◽  
Vol 104 (2) ◽  
pp. 380-389 ◽  
Author(s):  
Leon G. Coleman ◽  
Renata K. Polanowska-Grabowska ◽  
Marek Marcinkiewicz ◽  
Adrian R. L. Gear
Keyword(s):  
Platelet Aggregation ◽  
Protein Kinase ◽  
P38 Mapk ◽  
Platelet Function ◽  
Hypochlorous Acid ◽  
Plaque Rupture ◽  
Thrombus Formation ◽  
Mitogen Activated Protein Kinase ◽  
Low Levels

Abstract The in vitro oxidation of low-density lipoprotein (LDL) by hypochlorous acid produces a modified form (HOCl-LDL) capable of stimulating platelet function. We now report that HOCl-LDL is highly effective at inducing platelet function, causing stable aggregation and α-granule secretion. Such stimulation depended on the presence of low levels of primary agonists such as adenosine diphosphate (ADP) and thrombin, or others like epinephrine (EPI) and macrophage-derived chemokine (MDC, CCL22). Agonist levels, which by themselves induced little or reversible aggregation, caused strong stable aggregation when combined with low levels of HOCl-LDL. Platelet activation by HOCl-LDL and ADP (1 μM) caused P-selectin (CD62P) exposure, without serotonin or adenosine triphosphate (ATP) secretion. Intracellular calcium levels rose slowly (from 100 to 200 nM) in response to HOCl-LDL alone and rapidly when combined with ADP to about 300 nM. p38 mitogen-activated protein kinase (MAPK) became phosphorylated in response to HOCl-LDL alone. This phosphorylation was not blocked by the protein kinase C (PKC) inhibitor bisindolylmaleimide, which reduced the extent of aggregation and calcium increase. However, the p38 MAPK inhibitor SB203580 blocked platelet aggregation and phosphorylation of p38 MAPK. These findings suggest that HOCl-LDL exposed during atherosclerotic plaque rupture, coupled with low levels of primary agonists, can rapidly induce extensive and stable thrombus formation. (Blood. 2004;104:380-389)

Enhanced platelet MRP4 expression and correlation with platelet function in patients under chronic aspirin treatment

2016 ◽  
Vol 116 (12) ◽  
pp. 1100-1110 ◽  
Author(s):  
Isabella Massimi ◽  
Lavinia Lotti ◽  
Flavia Temperilli ◽  
Massimo Mancone ◽  
Gennaro Sardella ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Platelet Reactivity ◽  
Thrombus Formation ◽  
Independent Mechanism ◽  
Mrna And Protein Expression ◽  
Low Levels ◽  
Cox 1

SummaryPlatelet multidrug resistance protein4 (MRP4)-overexpression has a role in reducing aspirin action. Aspirin in vivo treatment enhances platelet MRP4 expression and MRP4 mediated transport inhibition reduces platelet function and delays thrombus formation. The aim of our work was to verify whether MRP4 expression is enhanced in platelets obtained from patients under chronic aspirin treatment and whether it correlates with residual platelet reactivity. We evaluated changes on mRNA and protein-MRP4 expression and platelet aggregation in four populations: healthy volunteers (HV), aspirin-free control population (CTR), patients who started the treatment less than one month ago (ASA<1 month patients) and aspirinated patients who started the treatment more than two months ago (ASA>2 months patients). In platelets obtained from ASA>2 months patients, it was found a statistically significant MRP4 enhancement of both mRNA and protein expression compared to HV, CTR and ASA<1 month patients. Platelets obtained from ASA>2 months patients that present high levels of platelet MRP4, have higher serum TxB2 levels and collagen-induced platelet aggregation compared to patient with low levels of MRP4 in platelets. In addition collagen induced platelet aggregation is higher in in vitro aspirinated platelets obtained from patients with high levels of MRP4 patients compared to those obtained from patients with low MRP4 levels. We can assert that, in patients under chronic aspirin treatment, platelets that present high MRP4 levels have an increase of residual platelet reactivity, which is due in part to incomplete COX-1 inhibition, and in part to COX-1–independent mechanism.

A novel vasculo-angiogenic effect of cilostazol mediated by cross-talk between multiple signalling pathways including the ERK/p38 MAPK signalling transduction cascade

2012 ◽  
Vol 123 (3) ◽  
pp. 147-159 ◽  
Author(s):  
Ting-Hsing Chao ◽  
Shih-Ya Tseng ◽  
Yi-Heng Li ◽  
Ping-Yen Liu ◽  
Chung-Lung Cho ◽  
...  
Keyword(s):  
Protein Kinase ◽  
P38 Mapk ◽  
Umbilical Vein ◽  
Tube Formation ◽  
Mitogen Activated Protein Kinase ◽  
Signalling Pathways ◽  
No Production ◽  
Angiogenic Effect

Cilostazol is an anti-platelet agent with vasodilatory activity that acts by increasing intracellular concentrations of cAMP. Recent reports have suggested that cilostazol may promote angiogenesis. In the present study, we have investigated the effect of cilostazol in promoting angiogenesis and vasculogenesis in a hindlimb ischaemia model and have also examined its potential mechanism of action in vitro and in vivo. We found that cilostazol treatment significantly increased colony formation by human early EPCs (endothelial progenitor cells) through a mechanism involving the activation of cAMP/PKA (protein kinase A), PI3K (phosphoinositide 3-kinase)/Akt/eNOS (endothelial NO synthase) and ERK (extracellular-signal-regulated kinase)/p38 MAPK (mitogen-activated protein kinase) signalling pathways. Cilostazol also enhanced proliferation, chemotaxis, NO production and vascular tube formation in HUVECs (human umbilical vein endothelial cells) through activation of multiple signalling pathways downstream of PI3K/Akt/eNOS. Cilostazol up-regulated VEGF (vascular endothelial growth factor)-A165 expression and secretion of VEGF-A in HUVECs through activation of the PI3K/Akt/eNOS pathway. In a mouse hindlimb ischaemia model, recovery of blood flow ratio (ipsilateral/contralateral) 14 days after surgery was significantly improved in cilostazol-treated mice (10 mg/kg of body weight) compared with vehicle-treated controls (0.63±0.07 and 0.43±0.05 respectively, P<0.05). Circulating CD34+ cells were also increased in cilostazol-treated mice (3614±670 compared with 2151±608 cells/ml, P<0.05). Expression of VEGF and phosphorylation of PI3K/Akt/eNOS and ERK/p38 MAPK in ischaemic muscles were significantly enhanced by cilostazol. Our data suggest that cilostazol produces a vasculo-angiogenic effect by up-regulating a broad signalling network that includes the ERK/p38 MAPK, VEGF-A165, PI3K/Akt/eNOS and cAMP/PKA pathways.

scholarly journals Cross-talk between IRAK-4 and the NADPH oxidase1

2007 ◽  
Vol 403 (3) ◽  
pp. 451-461 ◽  
Author(s):  
Sandrine Pacquelet ◽  
Jennifer L. Johnson ◽  
Beverly A. Ellis ◽  
Agnieszka A. Brzezinska ◽  
William S. Lane ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Nadph Oxidase ◽  
P38 Mapk ◽  
Interleukin 1 ◽  
Mitogen Activated Protein Kinase ◽  
Free System ◽  
Mitogen Activated Protein ◽  
A Cell ◽  
Tlr4 Activation

Exposure of neutrophils to LPS (lipopolysaccharide) triggers their oxidative response. However, the relationship between the signalling downstream of TLR4 (Toll-like receptor 4) after LPS stimulation and the activation of the oxidase remains elusive. Phosphorylation of the cytosolic factor p47phox is essential for activation of the NADPH oxidase. In the present study, we examined the hypothesis that IRAK-4 (interleukin-1 receptor-associated kinase-4), the main regulatory kinase downstream of TLR4 activation, regulates the NADPH oxidase through phosphorylation of p47phox. We show that p47phox is a substrate for IRAK-4. Unlike PKC (protein kinase C), IRAK-4 phosphorylates p47phox not only at serine residues, but also at threonine residues. Target residues were identified by tandem MS, revealing a novel threonine-rich regulatory domain. We also show that p47phox is phosphorylated in granulocytes in response to LPS stimulation. LPS-dependent phosphorylation of p47phox was enhanced by the inhibition of p38 MAPK (mitogen-activated protein kinase), confirming that the kinase operates upstream of p38 MAPK. IRAK-4-phosphorylated p47phox activated the NADPH oxidase in a cell-free system, and IRAK-4 overexpression increased NADPH oxidase activity in response to LPS. We have shown that endogenous IRAK-4 interacts with p47phox and they co-localize at the plasma membrane after LPS stimulation, using immunoprecipitation assays and immunofluorescence microscopy respectively. IRAK-4 was activated in neutrophils in response to LPS stimulation. We found that Thr133, Ser288 and Thr356, targets for IRAK-4 phosphorylation in vitro, are also phosphorylated in endogenous p47phox after LPS stimulation. We conclude that IRAK-4 phosphorylates p47phox and regulates NADPH oxidase activation after LPS stimulation.

Phosphorylation of Argonaute 2 at serine-387 facilitates its localization to processing bodies

2008 ◽  
Vol 413 (3) ◽  
pp. 429-436 ◽  
Author(s):  
Yan Zeng ◽  
Heidi Sankala ◽  
Xiaoxiao Zhang ◽  
Paul R. Graves
Keyword(s):  
Protein Kinase ◽  
P38 Mapk ◽  
Kinase Inhibitor ◽  
Mapk Pathway ◽  
Phosphorylation Site ◽  
Mitogen Activated Protein Kinase ◽  
Processing Bodies ◽  
Mitogen Activated Protein

Ago (Argonaute) proteins are essential effectors of RNA-mediated gene silencing. To explore potential regulatory mechanisms for Ago proteins, we examined the phosphorylation of human Ago2. We identified serine-387 as the major Ago2 phosphorylation site in vivo. Phosphorylation of Ago2 at serine-387 was significantly induced by treatment with sodium arsenite or anisomycin, and arsenite-induced phosphorylation was inhibited by a p38 MAPK (mitogen-activated protein kinase) inhibitor, but not by inhibitors of JNK (c-Jun N-terminal kinase) or MEK [MAPK/ERK (extracellular-signal-regulated kinase) kinase]. MAPKAPK2 (MAPK-activated protein kinase-2) phosphorylated bacterially expressed full-length human Ago2 at serine-387 in vitro, but not the S387A mutant. Finally, mutation of serine-387 to an alanine residue or treatment of cells with a p38 MAPK inhibitor reduced the localization of Ago2 to processing bodies. These results suggest a potential regulatory mechanism for RNA silencing acting through Ago2 serine-387 phosphorylation mediated by the p38 MAPK pathway.

C57BL/6JOlaHsd Mice with Tandem Deletion of the Multimerin 1 and Alpha-Synuclein Genes Have Impaired Platelet Function in Vivo and in Vitro That Can Be Corrected by Multimerin 1

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3926-3926 ◽  
Author(s):  
Subia Tasneem ◽  
Adili Reheman ◽  
Heyu Ni ◽  
Catherine P.M. Hayward
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Knockout Mice ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Alpha Synuclein ◽  
Type I ◽  
Significant Difference

Abstract Studies of mice with genetic deficiencies have provided important insights on the functions of many proteins in thrombosis and hemostasis. Recently, a strain of mice (C57BL/6JOlaHsd, an inbred strain of C57BL/6J) has been identified to have a spontaneous, tandem deletion of the multimerin 1 and α-synuclein genes, which are also adjacent genes on human chromosome 4q22. Multimerin 1 is an adhesive protein found in platelets and endothelial cells while α-synuclein is a protein found in the brain and in blood that is implicated in neurodegenerative diseases and exocytosis. In vitro, multimerin 1 supports platelet adhesion while α-synuclein inhibits α-granule release. We postulated that the loss of multimerin 1 and α-synuclein would alter platelet function and that recombinant human multimerin 1 might correct some of these abnormalities. We compared platelet adhesion, aggregation and thrombus formation in vitro and in vivo in C57BL/6JOlaHsd and C57BL/6 mice. Thrombus formation was studied by using the ferric-chloride injured mesenteric arteriole thrombosis model under intravital microscopy. We found that platelet adhesion, aggregation and thrombus formation in C57BL/6JOlaHsd were significantly impaired in comparison to control, C57BL/6 mice. The number of single platelets, deposited 3–5 minutes after injury, was significantly decreased in C57BL/6JOlaHsd mice (P <0.05, platelets/min: C57BL/6 = 157 ± 15, n=16; C57BL/6JOlaHsd = 77 ± 13, n=17). Moreover, thrombus formation in these mice was significantly delayed. Thrombi in C57BL/6JOlaHsd were unstable and easily dissolved, which resulted in significant delays (P<0.001) in vessel occlusion (mean occlusion times: C57BL/6 = 15.6 ± 1.2 min, n=16; C57BL/6JOlaHsd = 31.9 ± 2.1 min, n=17). We further tested platelet function in these mice by ADP and thrombin induced platelet aggregation using platelet rich plasma and gel-filtered platelets, respectively. Although no significant differences were seen with ADP aggregation, thrombin-induced platelet aggregation was significantly impaired in C57BL/6JOlaHsd mice. Platelet adhesion to type I collagen (evaluated using microcapillary chambers, perfused at 1500 s−1 with whole blood) was also impaired in C57BL/6JOlaHsd mice. However, platelets from C57BL/6JOlaHsd mice showed a normal pattern of agonist-induced release of α-granule P-selectin. Multimerin 1 corrected the in vitro aggregation and adhesion defects of C57BL/6JOlaHsd platelets. Furthermore, the transfusion of multimerin 1 into C57BL/6JOlaHsd mice corrected the impaired platelet deposition and thrombus formation in vivo. No significant difference was found in tail bleeding time between the two groups of mice. As α-synuclein knockout mice have a shortened time to thrombus formation (Circulation2007;116:II_76), the effects of multimerin 1 on impaired platelet function in C57BL/6JOlaHsd mice provide supportive evidence that multimerin 1 contributes to platelet adhesion and thrombus formation at the site of vessel injury. The findings suggest multimerin 1 knockout mice will be useful to explore platelet function. The first two authors and participating laboratories contributed equally to this study.

Novel 12-LOX Inhibitor ML355 Attenuates Platelet Reactivity and Impairs Thrombus Growth, Stability and Vessel Occlusion In Vivo

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3442-3442 ◽  
Author(s):  
Reheman Adili ◽  
Theodore R Holman ◽  
Michael Holinstat
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Platelet Adhesion ◽  
Ex Vivo ◽  
Platelet Reactivity ◽  
Thrombus Formation ◽  
Vessel Occlusion ◽  
Growth Stability

Abstract Background: Adequate platelet reactivity is required for platelet adhesion and aggregation at the site of vascular injury to maintain hemostasis. However, excessive platelet reactivity can also lead to the formation of occlusive thrombi, the predominate underlying cause of myocardial infarction and stroke. While current anti-platelet treatments limit platelet function, they often result in an increased risk of bleeding. 12-lipoxygenase (12-LOX), an oxygenase highly expressed in the platelet, has been demonstrated by our lab and others to regulate PAR4 and GPVI-mediated platelet reactivity suggesting a role of 12-LOX in regulation of vivo thrombosis. However, the ability to pharmacologically target 12-LOX in vivo has not been established to date. Aims: To determine how 12-LOX regulates thrombus formation in vivo and whether platelet 12-LOX is an effective target for anti-platelet therapeutics, wild-type (WT) or 12-LOX deficient (12-LOX-/-) mice were treated with or without the 12-LOX inhibitor, ML355, and were assessed for inhibitory effects on platelet activation in vitro, ex-vivo and in vivo. Methods: The effect of the novel 12-LOX inhibitor ML355 on human platelet function was assessed in vitro by platelet aggregometry, ex vivo by perfusion chamber. In vivo thrombus formation and vessel occlusion in small and large vessels were studied in 12-LOX-/-, WT mice and mice treated with ML355 using intravital microscopy using the FeCl3 injury models. Results: Using in vitro platelet aggregation assays, ML355 dose dependently inhibited thrombin, PAR1-AP, and PAR4-AP-induced aggregation in washed human platelets. Interestingly, the negative regulatory effects of ML355 inhibition of 12-LOX can be overcome by high concentration of thrombin. Additionally, ML355 was able to attenuate ADP-induced platelet aggregation both in platelet-rich-plasma and whole blood. In ex vivo flow chamber assays, platelet adhesion and thrombus formation on collagen-coated surfaces at high shear was attenuated in both mouse and human whole blood after incubation with ML355. Further, platelet aggregation and thrombus growth in 12-LOX-/- mice was impaired in FeCl3-induced mesenteric or carotid artery thrombosis models. Thrombi in 12-LOX-/- mice were unstable and frequently form emboli, which resulted in impaired vessel occlusion or reopening. Additionally, thrombus formation and vessel occlusion was impaired in ML355 treated WT mice. Conclusions: The highly selective 12-LOX inhibitor ML355 inhibits platelets aggregation induced by various platelet agonists and ML355 inhibition of platelet function is not agonist specific. Platelet function at high shear in ex vivo conditions in both mice and human was attenuated in the presence of ML355. Thrombus growth, stability, and vessel occlusion was impaired in mice deficient for 12-LOX. Finally, the highly selective 12-LOX inhibitor ML355 attenuates thrombus formation and prevents vessel occlusion in vivo. Our data strongly indicates 12- LOX is an important determinant of platelet reactivity and inhibition of platelet 12-LOX may represent a new target for anti-platelet therapeutics. Disclosures No relevant conflicts of interest to declare.

GLUT4 translocation precedes the stimulation of glucose uptake by insulin in muscle cells: potential activation of GLUT4 via p38 mitogen-activated protein kinase

2001 ◽  
Vol 359 (3) ◽  
pp. 639-649 ◽  
Author(s):  
Romel SOMWAR ◽  
David Y. KIM ◽  
Gary SWEENEY ◽  
Carol HUANG ◽  
Wenyan NIU ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Glucose Uptake ◽  
P38 Mapk ◽  
Mitogen Activated Protein Kinase ◽  
Kinase Assay ◽  
Glut4 Translocation ◽  
L6 Myotubes ◽  
Mitogen Activated Protein ◽  
Stimulation Of

We previously reported that SB203580, an inhibitor of p38 mitogen-activated protein kinase (p38 MAPK), attenuates insulin-stimulated glucose uptake without altering GLUT4 translocation. These results suggested that insulin might activate GLUT4 via a p38 MAPK-dependent pathway. Here we explore this hypothesis by temporal and kinetic analyses of the stimulation of GLUT4 translocation, glucose uptake and activation of p38 MAPK isoforms by insulin. In L6 myotubes stably expressing GLUT4 with an exofacial Myc epitope, we found that GLUT4 translocation (t1/2 = 2.5min) preceded the stimulation of 2-deoxyglucose uptake (t1/2 = 6min). This segregation of glucose uptake from GLUT4 translocation became more apparent when the two parameters were measured at 22°C. Preincubation with the p38 MAPK inhibitors SB202190 and SB203580 reduced insulin-stimulated transport of either 2-deoxyglucose or 3-O-methylglucose by 40–60%. Pretreatment with SB203580 lowered the apparent transport Vmax of insulin-mediated 2-deoxyglucose and 3-O-methylglucose without any significant change in the apparent Km for either hexose. The IC50 values for the partial inhibition of 2-deoxyglucose uptake by SB202190 and SB203580 were 1 and 2μM respectively, and correlated with the IC50 for full inhibition of p38 MAPK by the two inhibitors in myotubes (2 and 1.4μM, respectively). Insulin caused a dose- (EC50 = 15nM) and time- (t1/2 = 3min) dependent increase in p38 MAPK phosphorylation, which peaked at 10min (2.3±0.3-fold). In vitro kinase assay of immunoprecipitates from insulin-stimulated myotubes showed activation of p38α (2.6±0.3-fold) and p38β (2.3±0.2-fold) MAPK. These results suggest that activation of GLUT4 follows GLUT4 translocation and that both mechanisms contribute to the full stimulation of glucose uptake by insulin. Furthermore, activation of GLUT4 may occur via an SB203580-sensitive pathway, possibly involving p38 MAPK.

Levo-corydalmine attenuates microglia activation and neuropathic pain by suppressing ASK1-p38 MAPK/NF-κB signaling pathways in rat spinal cord

2020 ◽  
Vol 45 (3) ◽  
pp. 219-229 ◽  
Author(s):  
Wen-Ling Dai ◽  
Yi-Ni Bao ◽  
Ji-Fa Fan ◽  
Shan-Shan Li ◽  
Wan-Li Zhao ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Neuropathic Pain ◽  
Protein Kinase ◽  
P38 Mapk ◽  
Calcium Binding ◽  
Microglia Activation ◽  
Mitogen Activated Protein Kinase ◽  
Intragastric Administration ◽  
Tnf Α

Background and objectivesNeuropathic pain is partially refractory to currently available treatments. Although some studies have reported that apoptosis signal-regulating kinase 1 (ASK1) may inhibit chronic pain, the mechanisms underlying this process have not been fully elucidated.MethodsChronic constriction injury (CCI) of the rat sciatic nerve was used to establish a neuropathic pain model. Nociception was assessed using von Frey hair and Hargreaves’ methods. Western blot and immunofluorescence were used to detect the cell signaling pathway. BV2 cell line was cultured for in vitro evaluation.ResultsOur results indicated that spinal ASK1 was co-expressed with the microglia marker ionized calcium binding adaptor 1. Additionally, intrathecal administration of ASK1 inhibitor suppressed the activation of spinal microglia and attenuated CCI-induced neuropathic pain. The ASK1 inhibitor also decreased the levels of phosphorylated ASK1 (p-ASK1), p65, p38 mitogen-activated protein kinase (MAPK) and tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) messenger RNA in lipopolysaccharide-stimulated BV2 microglia cells. Intragastric administration of levo-corydalmine (l-CDL) significantly attenuated CCI-induced neuropathic pain and inhibited the expression of p-ASK1 in the spinal cord. l-CDL conspicuously suppressed the activation of spinal microglia in vitro and in vivo. Translocation of nuclearfactor-kappa B (NF-κB) and upregulation of p-p65, TNF-α, IL-1β were inhibited by l-CDL. Further, the analgesic effects of l-CDL were associated with reduced levels of phosphorylated protein kinase C (PKC γ), c-JunNH2-terminal kinase, and extracellular signal-regulated kinase.ConclusionsThis study showed that the expression of ASK1 in spinal microglia and ASK1 inhibitor suppressed microglia activation via suppression of p38 MAPK/NF-κB, which ultimately attenuated CCI-induced neuropathic pain. l-CDL also inhibited the ASK1-P38 MAPK/NF-κB axis to attenuate CCI-induced neuropathic pain.

Individual Variation in Hemostatic Alterations Caused By Tyrosine Kinase Inhibitors - a Way to Improve Personalized Cancer Therapy?

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1908-1908
Author(s):  
Kourosh Lotfi ◽  
Suryyani Deb ◽  
Clara Sjöström ◽  
Anjana Tharmakulanathan ◽  
Niklas Boknäs ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Side Effects ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Platelet Function ◽  
Thrombin Generation ◽  
Kinase Inhibitors ◽  
Thrombus Formation ◽  
Individual Variations

Abstract Introduction During the last two decades, Bcr-Abl tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of chronic myelogenous leukemia (CML), and are now considered standard treatment for this disease. However, TKIs can induce serious hemostatic side effects including cardiovascular disease and bleeding disorders. Blood platelet aggregation and formation of pro-coagulant platelets are important to allow a well-balanced hemostatic response. Therefore, a detailed understanding of what effect different TKIs exert on platelets and hemostasis could help to understand if there are differences of importance to minimize the risk of bleeding complications in treated patients. Aim To investigate how TKIs used in CML (imatinib, dasatinib, nilotinib, bosutinib, and ponatinib) affect platelet activation and hemostasis. Materials and Methods We have developed a multi-parameter six color flow cytometry protocol to study different aspects of platelet function upon activation, e.g. formation of aggregatory (PAC-1-positive) and pro-coagulant (phosphatidylserine-exposing) platelets, exocytosis of alpha- and lysosomal granules and mitochondrial membrane potential.This protocol was performed in presence or absence of TKIs in blood from normal donors and in treated patients. Whole blood aggregometry (Multiplate®), thrombin generation in platelet-rich plasma and in vitro thrombus formation by free oscillation rheometry (ReoRox G2) was further evaluated in some situations. Results At clinically relevant concentrations, dasatinib significantly decreased the formation of procoagulant platelets. Ponatinib induced a slight decrease in formation of procoagulant platelets, whereas bosutinib and nilotinib showed opposite tendencies (n=7). Dasatinib also decreased platelet aggregation (n=4-6) and in vitro thrombus formation (n=3). Thrombin generation was not significantly affected by therapeutic levels of TKIs, whereas higher doses of dasatinib, bosutinib, ponatinib and imatinib significantly changed one or several of the thrombin generation parameters (n=7-8). Interestingly, large differences in response to the drugs were observed among the healthy donors, especially for dasatinib and bosutinib. Major inter-individual variations were also observed in dasatinib-treated patients. Conclusions Different TKIs show varying potency to affect platelet-based hemostasis. In addition, we found large inter-individual variations in how some drugs affected platelet function. Therefore, we suggest that development of a clinically useful protocol for platelet function testing could help to identify patients more susceptible to adverse drug reactions. Such a protocol could potentially help clinicians to gain insight into the risk of side effects, which could help to choose the most suitable drug for each individual patient. Disclosures No relevant conflicts of interest to declare.

scholarly journals MSK1 activity is controlled by multiple phosphorylation sites

2005 ◽  
Vol 387 (2) ◽  
pp. 507-517 ◽  
Author(s):  
Claire E. McCOY ◽  
David G. CAMPBELL ◽  
Maria DEAK ◽  
Graham B. BLOOMBERG ◽  
J. Simon C. ARTHUR
Keyword(s):  
Protein Kinase ◽  
P38 Mapk ◽  
Kinase Domain ◽  
Mitogen Activated Protein Kinase ◽  
Mapk Cascades ◽  
Extracellular Signal ◽  
Mitogen Activated Protein ◽  
Ribosomal S6 Kinase ◽  
In Cells

MSK1 (mitogen- and stress-activated protein kinase) is a kinase activated in cells downstream of both the ERK1/2 (extracellular-signal-regulated kinase) and p38 MAPK (mitogen-activated protein kinase) cascades. In the present study, we show that, in addition to being phosphorylated on Thr-581 and Ser-360 by ERK1/2 or p38, MSK1 can autophosphorylate on at least six sites: Ser-212, Ser-376, Ser-381, Ser-750, Ser-752 and Ser-758. Of these sites, the N-terminal T-loop residue Ser-212 and the ‘hydrophobic motif’ Ser-376 are phosphorylated by the C-terminal kinase domain of MSK1, and their phosphorylation is essential for the catalytic activity of the N-terminal kinase domain of MSK1 and therefore for the phosphorylation of MSK1 substrates in vitro. Ser-381 is also phosphorylated by the C-terminal kinase domain, and mutation of Ser-381 decreases MSK1 activity, probably through the inhibition of Ser-376 phosphorylation. Ser-750, Ser-752 and Ser-758 are phosphorylated by the N-terminal kinase domain; however, their function is not known. The activation of MSK1 in cells therefore requires the activation of the ERK1/2 or p38 MAPK cascades and does not appear to require additional signalling inputs. This is in contrast with the closely related RSK (p90 ribosomal S6 kinase) proteins, whose activity requires phosphorylation by PDK1 (3-phosphoinositide-dependent protein kinase 1) in addition to phosphorylation by ERK1/2.

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