scholarly journals Apoptosis Signal-Regulating Kinase (ASK1) Regulates Thrombosis in Part By Regulating cPLA2 phosphorylation-Dependent TxA2 Generation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3719-3719
Author(s):  
Pravin Patel ◽  
Meghna U. Naik ◽  
Ulhas Naik
Keyword(s):  
Map Kinase ◽  
Platelet Activation ◽  
Platelet Function ◽  
Map Kinases ◽  
Conflicts Of Interest ◽  
Cellular Stress ◽  
Substantial Reduction ◽  
Genetic Ablation ◽  
Independent Mechanism

Abstract When vascular endothelium is injured, circulating platelets are activated by primary agonists. Activation causes platelets to change shape, aggregate, and release secondary agonists which reinforce initial platelet activation as well as help recruit additional platelets to the site of vascular injury. MAP kinases have been shown to be important regulators of platelet function and secondary agonist production. One important secondary agonist released by activated platelets is TxA2. TxA2 is generated by metabolism of Arachidonic acid (AA). AA is released from platelet membrane phospholipids via the activity of PLAs. In platelets cPLA2 activity has been shown to be regulated by MAP kinases, however, the mechanisms which regulate platelet MAP kinase activity are not well understood. Our laboratory has identified that ASK1 (a Ser/Thr kinase of the MAP3K family) is present in both human and murine platelets and is activated by physiological agonists. ASK1 is known to be activated by a number of cellular stress response pathways. When challenged by cellular stress, ASK1 auto phosphorylates Thr845 on its activation loop, which is required for its ability to phosphorylate its substrates. Here we show that ASK1 regulates platelet function in part by regulating agonist-induced TxA2 generation. To determine the role of Ask1 in hemostasis and thrombosis, we evaluated in vivo thrombosis using carotid artery injury induced by 10% FeCl3 or pulmonary thromboembolism induced by injecting mixture of collagen/epinephrine. We found that genetic ablation of Ask1 renders mice significant protection from thrombosis. To determine the mechanism by which Ask1 regulates platelet activation leading to thrombosis, we evaluated the MAP kinase cascade using Ask1 null platelets. We found that genetic ablation of Ask1 blocked agonist-induced activation of the MAP2Ks (MKK3 and MKK4) in murine platelets. Since MKK3 can activate p38 and MKK4 can activate both p38 and JNK, we assessed MAPKs activation in murine platelets. When stimulated by various agonists, activation of p38 was entirely lost in Ask1 null platelets while activation of ERK1/2 and JNK remained unaffected indicating that Ask1 solely regulates p38 activity in platelets. Activity of p38 has been linked to agonist-induced generation of TxA2, an important contributing factor to thrombosis. We therefore evaluated agonist-induced production of TxA2 by measuring TxB2 (a stable metabolite of TxA2). We saw a substantial reduction (~50% in thrombin- and ~70% in convulxin-induced) production of TxA2 in Ask1 null platelets suggesting a separate Ask1 independent mechanism for TxA2 generation. Since TxA2 is a metabolite of AA, whose production in platelets is caused by cPLA2 enzymatic activity and cPLA2 activity is regulated by phosphorylation of its Ser505 residue by p38, we evaluated phosphorylation of cPLA2 (p-Ser505). We found that agonist-induced phosphorylation of cPLA2 (Ser505) was completely lost in Ask1 null platelets. Although in Ask1 null platelets cPLA2 phosphorylation (Ser505) is completely abolished, substantial amount (~50%) of TxA2 was generated in response to thrombin suggesting that there exists an Ask1 independent mechanism of activation of cPLA2. To rule out the possibility that an alternative PLA2 is responsible for the residual TxA2 production found in Ask1 null platelets, we evaluated agonist-induced TxA2 production in the presence of pyrrophenone, a cPLA2 specific inhibitor. Pretreatment with pyrrophenone completely abolished agonist-induced TxA2 production in murine as well as human platelets, suggesting that cPLA2 is solely responsible for the majority of agonist induced AA/TxA2 in platelets. In addition to its phosphorylation, it is documented that cPLA2 activity is also dependent on intracellular Ca2+, which facilitates translocation of cPLA2 to AA containing membranes. It is therefore possible that the remainder of TxA2 formed is dependent on Ca2+-dependent activity of cPLA2. Taken together these in vivo and in vitro results strongly suggest that ASK1 plays a key role in regulating thrombosis, in part, by regulating the signaling mechanisms involved in agonist-induced production of TxA2. Disclosures No relevant conflicts of interest to declare.

Agonist-Induced Thromboxane A2 Generation Requires Signaling Through Apoptosis Signal-Regulating Kinase 1 (ASK1): Genetic Ablation of Ask1 Severely Affects In Vivo Thrombosis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 187-187
Author(s):  
Meghna Ulhas Naik ◽  
Ramya Turaga ◽  
Hidinori Ichijo ◽  
Ulhas P Naik
Keyword(s):  
Oxidative Stress ◽  
Platelet Aggregation ◽  
Map Kinase ◽  
Map Kinases ◽  
Bleeding Time ◽  
Conflicts Of Interest ◽  
Serotonin Release ◽  
Genetic Ablation ◽  
Cpla2 Activation

Abstract Abstract 187 Oxidative stress is a major contributing factor for cardiovascular diseases. Apoptosis signal-regulating kinase 1 (ASK1) is a member of the MAP kinase-kinase-kinase family, which responds to a diverse array of cellular stresses including oxidative stress. Although its downstream signaling effectors, the JNK and p38 MAP kinases, have been shown to be present and activated in platelets, the presence of ASK1 is not known. Here we show that ASK1 is highly expressed in human platelets. During activation of platelets by a variety of physiological agonists, ASK1 is rapidly activated by phosphorylation of threonine (T845) in its activation loop. In order to determine the physiological significance of activation of ASK1 we used Ask1 knockout mouse. Genetic ablation of Ask1 significantly delayed tail-bleeding time (P=0.2×10−9). While all WT mice stopped bleeding with an average bleeding time of 100 s, the Ask1 null mice had an average bleeding time of 576 s, with 5 out of 9 mice that did not stop bleeding for up to 10 min. A carotid artery injury induced by 10% FeCl3 showed a significantly increased (P=0.0003) time of occlusion and unstable thrombus formation in Ask1 null mice. Furthermore, we found that loss of Ask1 renders significant protection to the mice from pulmonary thromboembolism induced by intravenous injection of a mixture of collagen and epinephrine as determined by increased survival and a lack of exclusion of Evans blue dye from the lungs. To determine the cause of such a severe defect in thrombosis, we evaluated ex vivo platelet functions. We found that low doses of agonist-induced platelet aggregation were significantly reduced in Ask1 null mice compared to WT mice. Defective aggregation was found to be due in part to impaired activation of fibrinogen receptor as indicated by reduced fibrinogen binding. When the ability of Ask1 platelets to release platelet granular contents was analyzed, both a- and d-granule secretion were significantly inhibited as assessed by P-selectin exposure and 14C-serotonin release, respectively. Interestingly, aspirin treatment had no effect on the amount of 14C-serotonin release from Ask1 null platelets, but reduced the amount in WT platelets, bringing it to the same level as that of Ask1 null platelets, suggesting that agonist-induced thromboxane generation was impaired in the Ask1 null platelets. This was further confirmed by the complete lack of thrombin-induced TxA2 production in Ask1 null platelets as compared to WT. Since cPLA2 is a key enzyme in the pathway of TxA2 generation, we analyzed the effect of the absence of Ask1 on cPLA2 activation. We found a complete absence of cPLA2 activation-induced by thrombin in Ask1 null platelets while a robust activation of cPLA2 occurred in WT. Additionally, agonist-induced activation of the p38 MAP kinase, known to be involved in cPLA2 activation, was absent in Ask1 null platelets. Furthermore, the TxA2 mimetic U46619-induced platelet aggregation was also greatly reduced in Ask1 null platelets, suggesting that signaling through the TxA2 receptor is also impaired in Ask1 null platelets. These in vitro and in vivo results strongly suggest that ASK1 plays a major role in the regulation of hemostasis and thrombosis by regulating a number of signaling events that are critical for proper platelet function. Disclosures: No relevant conflicts of interest to declare.

Apoptosis Signal-Regulating Kinase 1 Regulates Platelet Function through Integrin aIIbb3 Outside-In Signaling

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 328-328
Author(s):  
Meghna Ulhas Naik ◽  
Hidinori Ichijo ◽  
Ulhas P Naik
Keyword(s):  
Map Kinase ◽  
Platelet Function ◽  
Stress Responses ◽  
Intracellular Signaling ◽  
Bleeding Time ◽  
Conflicts Of Interest ◽  
Phosphorylation Site ◽  
Mapk Cascades ◽  
Induced Aggregation

Abstract Abstract 328 Atherosclerosis tends to develop in areas of the circulation where the blood flow is either low or turbulent, such as regions where the arteries bifurcate. Studies have shown that shear stress stimulates endothelial cells and modulates platelet function. However, the intracellular signaling events that coordinate these effects are not completely understood. The mitogen-activated protein kinase (MAPK) cascades are crucial in regulating cellular stress responses. It is known that a number of diseases, including cardiovascular diseases, are intimately related to stress related mechanisms mediated by MAPK cascades. Apoptosis signal-regulating kinase 1 (ASK1) is a member of the MAP kinase-kinase-kinase family which responses to diverse array of stresses. Here, we show for the first time that ASK1, a 155 kDa protein, is present in human and murine platelets and its role as a novel regulator of platelet function. In human platelets, when stimulated with thrombin, ASK1 is rapidly activated within 30 sec, as indicated by phosphorylation of Thr 845 on its activation loop. Interestingly, we found that ASK1 is rapidly inactivated after 3 min by dephosphorylation of phospho-Thr 845. Concomitantly, ASK1 is phosphorylated on Ser 967, a known inhibitory phosphorylation site. The activation of ASK1 coincided well with its downstream effector, p38 Map kinase. These results suggested that ASK1 may play a role in platelet function. We therefore tested the hemostatic functions in congenic Ask-1 knockout mice and compared it to C57/BL6 wild type (Wt) mice. We found a significant delay in tail bleeding time (P=0.2×10−9) in Ask1 null mice. While all Wt mice stopped bleeding with an average bleeding time of 100 sec, the Ask1 null mice had an average bleeding time of 576 sec, with 5 out of 9 mice that did not stop bleeding. A 10% FeCl3-induced carotid artery injury, a well-established in vivo thrombosis model, showed a significantly increased (P=0.0003) time of occlusion and unstable thrombus formation in Ask1 null mice. Furthermore, we found that loss of Ask1 renders significant protection to the mice from pulmonary thromboembolism induced by intravenous injection of a mixture of collagen and epinephrine. This finding was further supported by the histological examination of the lung tissue of mice surviving after 3 min of injection, which showed very few thrombi in Ask1 null mice compared to Wt mice. We next asked if the platelet function is affected in Ask1 null mice. We found that low dose of PAR-4 peptide-induced aggregation is significantly less in Ask1 null mice compared to Wt mice. When analyzed for the ability of Ask1 null platelets to retract clots, we found that while clot retraction was completed within 2h in Wt platelets, Ask1 null platelets failed to retract clots even after 18h. When assessed for phosphorylation of the integrin b3 subunit as an indicator of outside-in signaling, we found that Wt platelet exposure to immobilized fibrinogen showed a robust phosphorylation of b3 subunit. Interestingly, Ask1 null platelets failed to show any induction of b3 phosphorylation. These in vitro and in vivo results strongly suggest that ASK1 plays a significant role in the regulation of platelet function and hemostasis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Nix-mediated mitophagy regulates platelet activation and life span

2019 ◽  
Vol 3 (15) ◽  
pp. 2342-2354 ◽  
Author(s):  
Weilin Zhang ◽  
Qi Ma ◽  
Sami Siraj ◽  
Paul A. Ney ◽  
Junling Liu ◽  
...  
Keyword(s):  
Life Span ◽  
Platelet Activation ◽  
Platelet Function ◽  
Bone Marrow Cells ◽  
Mitochondrial Protein ◽  
Genetic Ablation ◽  
Underlying Mechanisms ◽  
Adenosine Triphosphate Production ◽  
And Control

Abstract Platelet activation requires fully functional mitochondria, which provide a vital energy source and control the life span of platelets. Previous reports have shown that both general autophagy and selective mitophagy are critical for platelet function. However, the underlying mechanisms remain incompletely understood. Here, we show that Nix, a previously characterized mitophagy receptor that plays a role in red blood cell maturation, also mediates mitophagy in platelets. Genetic ablation of Nix impairs mitochondrial quality, platelet activation, and FeCl3-induced carotid arterial thrombosis without affecting the expression of platelet glycoproteins (GPs) such as GPIb, GPVI, and αIIbβ3. Metabolic analysis revealed decreased mitochondrial membrane potential, enhanced mitochondrial reactive oxygen species level, diminished oxygen consumption rate, and compromised adenosine triphosphate production in Nix−/− platelets. Transplantation of wild-type (WT) bone marrow cells or transfusion of WT platelets into Nix-deficient mice rescued defects in platelet function and thrombosis, suggesting a platelet-autonomous role (acting on platelets, but not other cells) of Nix in platelet activation. Interestingly, loss of Nix increases the life span of platelets in vivo, likely through preventing autophagic degradation of the mitochondrial protein Bcl-xL. Collectively, our findings reveal a novel mechanistic link between Nix-mediated mitophagy, platelet life span, and platelet physiopathology. Our work suggests that targeting platelet mitophagy Nix might provide new antithrombotic strategies.

Racial Differences In Thrombin-Induced Human Platelet PAR4 Reactivity

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1054-1054
Author(s):  
Leonard C. Edelstein ◽  
Lukas M Simon ◽  
Raul Teruel Montoya ◽  
Michael Holinstat ◽  
Edward Chen ◽  
...  
Keyword(s):  
Racial Differences ◽  
Platelet Activation ◽  
Platelet Function ◽  
Racial Difference ◽  
Conflicts Of Interest ◽  
Platelet Reactivity ◽  
Wild Type Mouse ◽  
Induced Aggregation ◽  
Megakaryocytic Cell

Abstract Compared to white patients, black patients have worse outcomes after acute coronary events, but there is a paucity of literature considering racial differences in platelet function. Thrombin is an especially potent in vivo platelet agonist, and no work has considered racial differences in thrombin-induced platelet aggregation. Using PAR1- and PAR4- activation peptides (APs), we recently reported that platelets from healthy black subjects (n = 70) demonstrated greater aggregation to the PAR4-AP than platelets from white subjects (n = 84) (p = 5.15 x 10-8). There was no racial difference to PAR1-AP, ADP or CRP. The goal of the current study was to determine if this racial difference in PAR4-AP-mediated platelet reactivity was also observed with thrombin and to investigate responsible molecular and genetic mechanism(s). A detailed dose-response study (4 blacks and 3 whites) revealed that when thrombin signaling was restricted to PAR4 by inhibiting PAR1 with BMS-200261, platelets from black subjects aggregated faster than platelets from white subjects at low concentrations of thrombin. No PAR1-AP-induced aggregation occurred in the presence of BMS-200261. A subsequent replication study (an additional 5 blacks and 5 whites) again showed platelets from black subjects aggregated faster than white subjects in the absence of PAR1 signaling (p = 3.56x10-5). DNA from all 154 subjects was genotyped for 5 million SNPs (HumanOmni5 array) and principal component analysis revealed that the genotypes segregated into two distinct groups that correlated perfectly with subject self-identified race. Gene expression profiling on leukocyte-depleted platelets from all 154 subjects revealed numerous differentially expressed (DE) RNAs associated with both race and PAR4 reactivity. The gene encoding phosphatidylcholine transfer protein (PC-TP), PCTP, showed the strongest correlation with race (p = 10-23; q = 10-20) and with PAR4 reactivity (p = 3.4x10-8; q = 3.5x10-4). PC-TP protein was higher in platelets from blacks (p = 3.8x10-6) and levels correlated with reactivity to PAR4-AP (r = 0.249, p = 0.002). Pctp has been knocked out in mice, but we found that wild type mouse platelets express little or no Pctp protein, consistent with mouse platelet RNA data from Rowley et al (Blood 2011). However, a specific PC-TP inhibitor resulted in a reduced aggregation response to PAR4-AP, but not PAR1-AP. Transfection of a siRNA against PCTP reduced both PCTP mRNA and PC-TP protein levels, and inhibited Ca+ release in a megakaryocytic cell line, Meg-01 in response to PAR4-AP but not PAR1-AP. A racial difference in platelet Ca+ release in response to PAR4-AP treatment was also observed (p = 0.02). Platelet microRNA (miRNA) profiling from all 154 subjects also revealed numerous DE miRNAs associated with both race and PAR4 reactivity. Target prediction analysis indicated that miR-376c is a candidate for regulating PCTP expression. qRT-PCR of all 154 subjects indicated that miR-376c levels are expressed higher in platelets from whites (p = 1.47 x10-4; q = 1.38 x10-3), and are inversely correlated with PCTP mRNA (r = -0.214; p = 0.008), PC-TP protein (r = -0.211; p = 0.009) and PAR4 reactivity (r = -0.161; p = 0.049). Transfection of megakaryocytic cell lines or cord blood CD34+ derived megakaryocytes with the pre-miR-376c precursor or LNA-miR-376c inhibitor resulted in decreased (P<0.01) or increased PCTP (p = 0.0003), respectively. Co-transfection of the miRNA precursor or inhibitor with a luciferase vector containing the PCTP 3’UTR indicated the regulation was dependent on the predicted miR-376c target site. In summary, we have uncovered a racial difference in thrombin-induced PAR4 platelet activation. This finding has potential clinical significance because PAR4 is the primary means by which thrombin activates platelets in the presence of vorapaxar (a PAR1 inhibitor in clinical trials), and the risks and benefits of vorapaxar by race are unknown. This racial difference in platelet activation is mediated, in part, by PC-TP, a novel protein in platelet biology. Our data also supports racial differences in miRNA expression, one of which (miR-376c) regulates PC-TP expression. These results indicate a genomic contribution to platelet function that differs by race, emphasize a need to consider race effects when developing anti-thrombotic drugs and raise the possibility that PC-TP inhibition might be a useful anti-thrombotic strategy. Disclosures: No relevant conflicts of interest to declare.

GPIb-dependent platelet activation is dependent on Src kinases but not MAP kinase or cGMP-dependent kinase

Blood ◽  
2004 ◽  
Vol 103 (7) ◽  
pp. 2601-2609 ◽  
Author(s):  
Stuart J. Marshall ◽  
Yotis A. Senis ◽  
Jocelyn M. Auger ◽  
Robert Feil ◽  
Franz Hofmann ◽  
...  
Keyword(s):  
Map Kinase ◽  
Platelet Activation ◽  
Map Kinases ◽  
Thrombus Formation ◽  
Critical Role ◽  
Rabbit Model ◽  
Guanosine Monophosphate ◽  
Src Kinases ◽  
Aggregate Formation ◽  
No Donor

Abstract Glycoprotein Ib-IX-V (GPIb-IX-V) mediates platelet tethering to von Willebrand factor (VWF), recruiting platelets into the thrombus, and activates integrin αIIbβ3 through a pathway that is dependent on Src kinases. In addition, recent reports indicate that activation of αIIbβ3 by VWF is dependent on protein kinase G (PKG) and mitogen-activated protein (MAP) kinases. The present study compares the importance of these signaling pathways in the activation of αIIbβ3 by GPIb-IX-V. In contrast to a recent report, VWF did not promote an increase in cyclic guanosine monophosphate (cGMP), while agents that elevate cGMP, such as the nitrous oxide (NO) donor glyco–SNAP-1 (N-(β-D-glucopyranosyl)-N2-acetyl-S-nitroso-D,L-penicillaminamide) or the type 5 phosphosdiesterase inhibitor, sildenafil, inhibited rather than promoted activation of αIIbβ3 by GPIb-IX-V and blocked aggregate formation on collagen at an intermediate rate of shear (800 s-1). Additionally, sildenafil increased blood flow in a rabbit model of thrombus formation in vivo. A novel inhibitor of the MAP kinase pathway, which is active in plasma, PD184161, had no effect on aggregate formation on collagen under flow conditions, whereas a novel inhibitor of Src kinases, which is also active in plasma, PD173952, blocked this response. These results demonstrate a critical role for Src kinases but not MAP kinases in VWF-dependent platelet activation and demonstrate an inhibitory role for cGMP-elevating agents in regulating this process.

Phosphorylation of caldesmon by ERK MAP kinases in smooth muscle

2000 ◽  
Vol 278 (4) ◽  
pp. C718-C726 ◽  
Author(s):  
Jason C. Hedges ◽  
Brian C. Oxhorn ◽  
Michael Carty ◽  
Leonard P. Adam ◽  
Ilia A. Yamboliev ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Map Kinase ◽  
Map Kinases ◽  
Isometric Force ◽  
Phosphorylation Site ◽  
Smooth Muscle Contraction ◽  
Tracheal Smooth Muscle ◽  
Muscarinic Agonists

Phosphorylation of h-caldesmon has been proposed to regulate airway smooth muscle contraction. Both extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinases phosphorylate h-caldesmon in vitro. To determine whether both enzymes phosphorylate caldesmon in vivo, phosphorylation-site-selective antibodies were used to assay phosphorylation of MAP kinase consensus sites. Stimulation of cultured tracheal smooth muscle cells with ACh or platelet-derived growth factor increased caldesmon phosphorylation at Ser789 by about twofold. Inhibiting ERK MAP kinase activation with 50 μM PD-98059 blocked agonist-induced caldesmon phosphorylation completely. Inhibiting p38 MAP kinases with 25 μM SB-203580 had no effect on ACh-induced caldesmon phosphorylation. Carbachol stimulation increased caldesmon phosphorylation at Ser789 in intact tracheal smooth muscle, which was blocked by the M2 antagonist AF-DX 116 (1 μM). AF-DX 116 inhibited carbachol-induced isometric contraction by 15 ± 1.4%, thus dissociating caldesmon phosphorylation from contraction. Activation of M2 receptors leads to activation of ERK MAP kinases and phosphorylation of caldesmon with little or no functional effect on isometric force. P38 MAP kinases are also activated by muscarinic agonists, but they do not phosphorylate caldesmon in vivo.

Activation of MAP kinases in airway smooth muscle

1997 ◽  
Vol 272 (2) ◽  
pp. L244-L252 ◽  
Author(s):  
W. T. Gerthoffer ◽  
I. A. Yamboliev ◽  
J. Pohl ◽  
R. Haynes ◽  
S. Dang ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Map Kinase ◽  
Airway Smooth Muscle ◽  
Map Kinases ◽  
Sodium Dodecyl ◽  
Western Blots ◽  
Mitogen Activated Protein ◽  
Intact Muscle ◽  
Triton X

To test the hypothesis that mitogen-activated protein (MAP) kinases are activated by contractile agonists in intact nonproliferating airway smooth muscle, kinase activities were compared in resting and stimulated canine tracheal smooth muscle. Kinase activities in sodium dodecyl sulfate extracts were assayed by a gel renaturation method. Myelin basic protein kinase activities corresponding to ERK1 and ERK2 immunoreactive proteins were activated twofold above the basal level within 5 min by 1 microM carbachol. MAP kinase activity assayed in crude homogenates using a synthetic peptide substrate (APRTPGGRR) also increased twofold above basal in muscles stimulated with 1 microM carbachol. Two protein kinases separated by Mono-Q chromatography were identified on Western blots as ERK1 and ERK2 MAP kinases. Carbachol stimulation increased caldesmon phosphorylation in intact muscle, and purified caldesmon was a substrate for activated murine ERK2 MAP kinase. Activated ERK2 MAP kinase added to Triton X-100-permeabilized fibers potentiated Ca2+-induced contraction. The results show that ERK MAP kinases are activated after stimulation of muscarinic receptors in airway smooth muscle, which is consistent with coupling of MAP kinases to phosphorylation of caldesmon in vivo.

Coupling of M2 muscarinic receptors to ERK MAP kinases and caldesmon phosphorylation in colonic smooth muscle

2000 ◽  
Vol 278 (3) ◽  
pp. G429-G437 ◽  
Author(s):  
Amy K. Cook ◽  
Michael Carty ◽  
Cherie A. Singer ◽  
Ilia A. Yamboliev ◽  
William T. Gerthoffer
Keyword(s):  
Smooth Muscle ◽  
Map Kinase ◽  
Muscarinic Receptors ◽  
Map Kinases ◽  
Kinase Inhibitor ◽  
Receptor Activation ◽  
Inhibitory Effect ◽  
Subsequent Treatment ◽  
P38 Map Kinase

Coupling of M2 and M3 muscarinic receptors to activation of mitogen-activated protein (MAP) kinases and phosphorylation of caldesmon was studied in canine colonic smooth muscle strips in which M3 receptors were selectively inactivated by N, N-dimethyl-4-piperidinyl diphenylacetate (4-DAMP) mustard (40 nM). ACh elicited activation of extracellular signal-regulated kinase (ERK) 1, ERK2, and p38 MAP kinases in control muscles and increased phosphorylation of caldesmon (Ser789), a putative downstream target of MAP kinases. Alkylation of M3 receptors with 4-DAMP had only a modest inhibitory effect on ERK activation, p38 MAP kinase activation, and caldesmon phosphorylation. Subsequent treatment with 1 μM AF-DX 116 completely prevented activation of ERK and p38 MAP kinase and prevented caldesmon phosphorylation. Caldesmon phosphorylation was blocked by the MAP kinase/ERK kinase inhibitor PD-98509 but not by the p38 MAP kinase inhibitor SB-203580. These results indicate that colonic smooth muscle M2 receptors are coupled to ERK and p38 MAP kinases. Activation of ERK, but not p38 MAP kinases, results in phosphorylation of caldesmon in vivo, which is a novel function for M2receptor activation in smooth muscle.

scholarly journals Activation of MAP kinase-activated protein kinase 2 in human neutrophils after phorbol ester or fMLP peptide stimulation

Blood ◽  
1996 ◽  
Vol 87 (12) ◽  
pp. 5287-5296 ◽  
Author(s):  
YL Zu ◽  
Y Ai ◽  
A Gilchrist ◽  
ME Labadia ◽  
RI Sha'afi ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Enzymatic Activity ◽  
Map Kinase ◽  
Phorbol Ester ◽  
Map Kinases ◽  
Phosphorylation Site ◽  
Human Neutrophils ◽  
Inhibitory Peptide

In response to extracellular stimulation, one of the earliest events in human neutrophils is protein phosphorylation, which mediates signal transduction and leads to the regulation of cellular functions. Mitogen- activated protein (MAP) kinases are rapidly activated by a variety of mitogens, cytokines, and stresses. The activated MAP kinases in turn regulate their substrate molecules by phosphorylation. MAP kinase- activated protein (MAPKAP) kinase 2, a Ser/Thr kinase, has been shown to be phosphorylated by p38 MAP kinase both in vivo and in vitro. Phosphorylation of the Thr-334 site of MAPKAP kinase 2 results in a conformational change with subsequent activation of the enzyme. To better define the role of MAPKAP kinase 2 in the activation of human neutrophils, its enzymatic activity was measured after stimulation by either a phorbol ester (phorbol myristate acetate [PMA]), a potent protein kinase C activator, or the tripeptide fMLP, which is a chemotactic factor. The in vitro kinase assays indicate that both PMA and fMLP stimulated a transient increase in the enzymatic activity of cellular MAPKAP kinase 2. The induced kinase activation was concentration-dependent and reached a maximum at 5 minutes for PMA and 1 minute for fMLP. To identify potential substrate molecules for MAPKAP kinase 2, a highly active kinase mutant was generated by mutating the MAP kinase phosphorylation site in the C-terminal region. The replacement of threonine 334 with alanine resulted in a marked augmentation of catalytic activity. Analysis of in vitro protein phosphorylation in the presence of the active kinase indicates that a 60-kD cytosolic protein (p60) was markedly phosphorylated and served as the major substrate for MAPKAP kinase 2 in human neutrophils. Based on the MAPKAP kinase 2 phosphorylation site of Hsp27, a competitive inhibitory peptide was synthesized. This competitive inhibitory peptide specifically inhibited MAPKAP kinase 2 enzymatic activity, as well as the in vitro and in vivo kinase-induced p60 phosphorylation. To assess the contribution of MAPKAP kinase 2 in neutrophil function, the oxidative burst response after manipulation of endogenous kinase activity was measured. Intracellular delivery of the competitive inhibitory peptide into human neutrophils reduced both PMA- and fMLP- stimulated superoxide anion production. Thus, the results strongly suggest that MAPKAP kinase 2 is involved in the activation of human neutrophils.

Understanding and Evaluating Platelet Function

Hematology ◽  
2010 ◽  
Vol 2010 (1) ◽  
pp. 387-396 ◽  
Author(s):  
Lawrence Brass
Keyword(s):  
Platelet Activation ◽  
Platelet Function ◽  
Tumor Angiogenesis ◽  
Clinical Setting ◽  
Molecular Level ◽  
Current Knowledge ◽  
Antiplatelet Agents ◽  
Normal Hemostasis ◽  
The Impact

Abstract The contribution of platelets to normal hemostasis and vascular disease is well described. However, recent studies make it clear that much remains to be learned about platelet activation at the single cell and the molecular level, and about the contribution of platelets to inflammation, tumor angiogenesis, and embryonic development. This article is divided into two themes. The first is an overview of current knowledge of the mechanisms that drive platelet function in vivo and a brief summary of some of the emerging ideas that are modifying older views. The second theme is a consideration of the strengths and weaknesses of the tools we have as hematologists to assess platelet function in the clinical setting, identify mechanisms, and evaluate the impact of antiplatelet agents.

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