scholarly journals Phorbol esters sensitize platelets to activation by physiological agonists

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1373-1381 ◽  
Author(s):  
W Siess ◽  
EG Lapetina
Keyword(s):  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Phorbol Esters ◽  
Platelet Rich Plasma ◽  
Dense Granule ◽  
Myosin Light Chain Phosphorylation ◽  
Kinase C

Phorbol esters such as phorbol 12, 13-dibutyrate (PdBu; 40 to 200 nmol/L) or 12-O-tetradecanoyl phorbol 13-acetate (20 to 80 nmol/L) added to aspirinized platelet-rich plasma (PRP) 5 to 15 seconds prior to various platelet stimuli (epinephrine, ADP, prostaglandin endoperoxide analog U44069, collagen, PAF, or vasopressin) potentiate the rate and extent of aggregation and ATP secretion induced by those agonists. Platelet aggregation, but not secretion, is potentiated at low concentrations of agonists; platelet secretion is potentiated at higher concentrations of the platelet stimuli. Potentiation of platelet responses was also observed when the preincubation time with PdBu was extended to 12 minutes and also occurred in washed platelets. The potentiating effect of phorbol esters is not mediated by formation of arachidonate metabolites or by released ADP. The sensitizing effect of PdBu on platelet aggregation induced by epinephrine is unique, since in contrast to the other platelet stimuli it is also found at maximal concentrations of epinephrine and does not diminish with prolonged preincubation of platelets with PdBu. Activation of protein kinase C ranges from 20% to 80% over control after 1 to 10 minutes of platelet pretreatment with PdBu but dramatically increases after subsequent addition of a stimulus such as vasopressin. In contrast, agonist- induced myosin light chain phosphorylation is reduced after platelet pretreatment with PdBu. The results indicate that protein kinase C activation enhances platelet aggregation and dense granule secretion triggered by physiologic stimuli, although it desensitizes agonist- induced myosin light chain phosphorylation.

scholarly journals Phorbol esters sensitize platelets to activation by physiological agonists

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1373-1381 ◽  
Author(s):  
W Siess ◽  
EG Lapetina
Keyword(s):  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Phorbol Esters ◽  
Platelet Rich Plasma ◽  
Dense Granule ◽  
Myosin Light Chain Phosphorylation ◽  
Kinase C

Abstract Phorbol esters such as phorbol 12, 13-dibutyrate (PdBu; 40 to 200 nmol/L) or 12-O-tetradecanoyl phorbol 13-acetate (20 to 80 nmol/L) added to aspirinized platelet-rich plasma (PRP) 5 to 15 seconds prior to various platelet stimuli (epinephrine, ADP, prostaglandin endoperoxide analog U44069, collagen, PAF, or vasopressin) potentiate the rate and extent of aggregation and ATP secretion induced by those agonists. Platelet aggregation, but not secretion, is potentiated at low concentrations of agonists; platelet secretion is potentiated at higher concentrations of the platelet stimuli. Potentiation of platelet responses was also observed when the preincubation time with PdBu was extended to 12 minutes and also occurred in washed platelets. The potentiating effect of phorbol esters is not mediated by formation of arachidonate metabolites or by released ADP. The sensitizing effect of PdBu on platelet aggregation induced by epinephrine is unique, since in contrast to the other platelet stimuli it is also found at maximal concentrations of epinephrine and does not diminish with prolonged preincubation of platelets with PdBu. Activation of protein kinase C ranges from 20% to 80% over control after 1 to 10 minutes of platelet pretreatment with PdBu but dramatically increases after subsequent addition of a stimulus such as vasopressin. In contrast, agonist- induced myosin light chain phosphorylation is reduced after platelet pretreatment with PdBu. The results indicate that protein kinase C activation enhances platelet aggregation and dense granule secretion triggered by physiologic stimuli, although it desensitizes agonist- induced myosin light chain phosphorylation.

Phorbol ester-induced contraction in chemically skinned vascular smooth muscle

1986 ◽  
Vol 251 (3) ◽  
pp. C356-C361 ◽  
Author(s):  
M. Chatterjee ◽  
M. Tejada
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Vascular Smooth Muscle ◽  
Phorbol Ester ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Phorbol Esters ◽  
Myosin Light Chain Phosphorylation ◽  
Kinase C

We studied the contractile response to phorbol esters and its relationship to myosin light chain phosphorylation in intact and Triton X-100-skinned porcine carotid preparations. Muscle contraction was activated by phorbol 12,13-dibutyrate (PDBu) and phorbol 12,13-didecanoate (PDD). Dose-dependent contractions to PDBu were obtained both in the intact and skinned preparations. The maximal values of stress in response to PDBu were 1.11 +/- 0.10 X 10(5) N/m2 (n = 7) in the intact and 5.72 +/- 0.59 X 10(4) N/m2 (n = 10) in the skinned muscles. The skinned tissues responded to PDD, which has been shown to activate protein kinase C, but not to the inactive isomer 4 alpha-PDD, thus ruling out nonspecific phorbol effects. The phorbol ester response exhibited a Ca2+ dependence. High stresses in the skinned muscles (5.53 +/- 0.69 X 10(4) N/m2, n = 8) were associated with low values of myosin light chain phosphorylation (0.18 +/- 0.01 mol Pi/mol light chain, n = 8). Thus phorbol esters can contract vascular smooth muscle by a mechanism that is not proportional to myosin light chain phosphorylation and that may involve activation of protein kinase C.

scholarly journals Isolation and characterization of protein kinase C from Y-1 adrenal cell cytoskeleton.

1989 ◽  
Vol 108 (2) ◽  
pp. 553-567 ◽  
Author(s):  
V Papadopoulos ◽  
P F Hall
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Phorbol Esters ◽  
Intact Cells ◽  
Adrenal Cells ◽  
Isolation And Characterization ◽  
Kinase C

The cytoskeletons of Y-1 mouse adrenal tumor cells contain a calcium and phospholipid-dependent protein kinase (protein kinase C) that is bound sufficiently tight to resist extraction by 0.5% Triton but not by 1.0% Triton. The enzyme has been purified to near homogeneity from cytoskeleton and cytosol. It shows features typical of this type of kinase, namely a requirement for Ca2+ and phospholipid, stimulation by tumor promoters but not by nontumor-promoting phorbol esters, and inhibition by trifluoperazine. The enzyme shows specificity for four substrates found in the cytoskeleton, namely 80, 33, 20, and 18 kD. The first three substrates are phosphorylated by the enzyme; the fourth is dephosphorylated and is therefore affected by the kinase indirectly. The 80-kD protein is the kinase enzyme itself which is autophosphorylated in vitro and in the cytoskeleton. The 20-kD protein is myosin light chain. The 33- and 18-kD proteins are unidentified. The same substrates were phosphorylated when Y-1 cells were permeabilized with digitonin and incubated with [gamma-32P]ATP and phorbol-12-myristate-13-acetate. Partly purified protein kinase C changes the extent of phosphorylation of the same substrates when added to cytoskeletons previously extracted to remove endogenous protein kinase C. Addition of Ca2+, phosphatidylserine, and phorbol-12-myristate-13-acetate to cytoskeletons, and addition of these three agents plus protein kinase C to extracted cytoskeletons, causes these structures to undergo a rapid and extensive rounding. A similar change is induced in intact cells by addition of phorbol ester. It is concluded that protein kinase C is capable of changing the shape of adrenal cells by an action that involves autophosphorylation and phosphorylation of myosin light chain. This response may in turn be related to the steroidogenic responses to ACTH and cyclic AMP.

SYNERGISM OF Gi-DISSOCIATION AND PROTEIN KINASE C STIMULATION IN PLATELET ACTIVATION

1987 ◽  
Author(s):  
W Siess ◽  
E G Lapetina
Keyword(s):  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Phosphatidic Acid ◽  
Protein Phosphorylation ◽  
Phospholipase C ◽  
Phorbol Esters ◽  
Platelet Rich Plasma ◽  
Kinase C ◽  
Α2 Adrenoceptor

Epinephrine or UK 14304 (a specific (α2-adrenoceptor agonist) synergizes with phorbol esters (phorbol 12,13-dibutyrate, PdBu) or bioactive diacylglycerols (sn-1,2-dioctanoylglycerol, DiC8) to induce aggregation and ATP-secretion of platelets. The effect on aggregation is more pronounced than on secretion, and it is observed in aspirinized platelet-rich plasma or suspensions of washed platelets containing ADP-scavengers. No prior shape change is found. In the presence of epinephrine, DiCg induces reversible aggregation and PdBu evokes irreversible aggregation that correlates with the effects, on protein phosphorylation. Epinephrine and UK 14304 neither induce nor enhance the phosphorylation of myosin light chain (20kDa), the substrate of protein kinase C (47kDa), or a 38kDa protein evoked by DiCg) or PdBu. Epinephrine does not cause, stimulation of phospholipase C as reflected by the production of inositol mono-, bis- and tris-phosphate or phosphatidic acid. Even under conditions of maximal aggregation induced by epinephrine plus PdBu, formation of 32p-phOSphatidic acid is not observed. The synergistic action of epinephrine and PdBu does not depend on extracellular Ca2+. Primary aggregation induced by epinephrine, but not platelet aggregation induced by PdBu plus epinephrine, is inhibited by high intracellular concentrations of the calcium chelator quin2. Prostacyclin prevents platelet aggregation but does not affect protein phosphorylation induced by PdBu plus epinephrine.The experiments indicate that α2-adrenoceptor agonists may induce primary aggregation by a mechanism involving release of membrane-bound Ca2+. The synergism with protein kinase C is, however, caused by a mechanism that occurs distally to protein phosphorylation and is not related to Phospholipase C activation and Ca2+-fluxes across the Dlasma membrane or in the cvtosol. Evidence is presented suoportina the view that this mechanism miqht be related to the dissociation of Gi caused by α2-adrenoceptor activation.

RUNX1 Regulates PLDN (Pallidin) Expression In Platelets: A Potential Mechanism For Dense Granule Deficiency In RUNX1 Haplodeficiency

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 567-567
Author(s):  
Guangfen Mao ◽  
Chengxiang Fan ◽  
Gauthami Jalagadugula ◽  
Robert Freishtat ◽  
A. Koneti Rao
Keyword(s):  
Transcription Factor ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Light Chain ◽  
Binding Sites ◽  
Myosin Light Chain ◽  
Normal Subjects ◽  
Dense Granule ◽  
Kinase C ◽  
Hel Cells

Abstract Transcription factor RUNX1 plays a major role in hematopoiesis. Haplodeficiency of RUNX1 is associated with familial thrombocytopenia, impaired platelet function and megakaryopoiesis, and predisposition to acute myeloid leukemia. Platelet abnormalities include impaired aggregation, secretion, protein phosphorylation, and activation of GPIIb-IIIa on platelet activation. Dense granule deficiency, either alone or in combination with alpha granule deficiency, has been reported in patients with RUNX1 haplodeficiency, but the mechanisms involved are unknown. We have reported a patient with inherited thrombocytopenia and abnormal platelet function (Gabbeta et al, Blood 87:1368-76, 1996) associated with a heterozygous nonsense mutation in transcription factor RUNX1 (Sun et al, Blood 103: 948-54, 2004). The platelets showed impaired aggregation, dense granule secretion, phosphorylation of pleckstrin and myosin light chain, diminished GPIIb-IIIa activation and decreased platelet protein kinase C-θ. Platelet mRNA expression profiling showed downregulation of several genes, including myosin light chain 9 (MYL9), platelet factor 4 (PF4), protein kinase C-θ (PRKB), and 12-lipoxygenase (ALOX12) in the patient compared to 6 normal subjects (Sun et al, J. Thromb Haemost. 5: 146-54, 2007). (We have shown that these genes are direct transcriptional targets of RUNX1.) In addition, PLDN, which encodes for protein pallidin, was four-fold down-regulated in platelets (fold change 0.239, p=0.029). In mouse model pallid, knock out of pldn leads to dense granule deficiency. Recent reports document mutations in PLDN in human subjects with platelet dense granule deficiency and the Hermansky-Pudlak syndrome. Pallidin is one of 8 subnunits that constitute the BLOC-1 (biogenesis of lysosome-related organelles complex-1), which plays a major role in granule/vesicle biogenesis. PLDN has two known human transcripts; PLDN1 is expressed ubiquitously, with exception of brain, while PLDN2 is expressed in brain, testes and leukocytes. We have addressed that PLDN is a direct transcriptional target of RUNX1 and hypothesize that its decreased expression constitutes a mechanism for the platelet dense granule deficiency in patients with RUNX1 haplodeficiency. We validated the decreased expression of PLDN on platelet profiling by quantitative real-time PCR - PLDN1 mRNA expression was decreased by 16-fold in the patient relative to normal subjects. We studied the regulation of PLDN1 by RUNX1 in human erythroleukemia (HEL) cells treated with phorbol 12-myristate 13-acetate (PMA) for 24 h to induce megakaryocytic transformation. In silico analyses revealed the presence of 6 RUNX1 consensus binding sites in 2288bp of PLDN1 5’ upstream region from ATG. To determine endogenous interaction of RUNX1 with PLDN promoter, we performed chromatin immunoprecipitation (ChIP) assay using anti-RUNX1 antibody. There was RUNX1 binding to PLDN chromatin at regions encompassing the putative RUNX1 binding site 1 (-184 to -179 bp) and site 3 (-1370/-1365 bp). We performed electrophoretic mobility shift assay (EMSA) using probes with RUNX1 motifs and PMA-treated HEL cell nuclear extracts. With 30–34 bp probes encompassing site 1 (-184 to -179 bp) and site 3 (-1370 to -1365 bp), protein binding was observed that was competed by excess unlabelled probe and inhibited by anti-RUNX1 antibody indicating RUNX1 as the protein involved. To study the functional effect of the binding sites, the wild type PLDN1 promoter construct –2288/-2 bp containing the putative RUNX1 sites or mutant constructs with each site individually mutated were cloned into firefly luciferase reporter gene vector and transfected into HEL cells. Mutation of RUNX1 sites 1 and 3 individually caused 60-70% reduction in promoter activity indicating that these sites were functional. Studies on the other RUNX1 consensus sites in PLDN promoter are underway. Conclusions Our results provide the first evidence that PLDN gene is transcriptionally regulated by and is a direct target of RUNX1. These studies provide a cogent mechanism for the PLDN transcript downregulation observed in the patient platelets. More importantly, they provide a mechanism for the dense granule deficiency and impaired vesicle formation associated with RUNX1 haplodeficiency. Disclosures: No relevant conflicts of interest to declare.

Differential Regulation of Myosin Regulatory Light Chain Phosphorylation by Protein Kinase C Isozymes in Human Uterine Myocytes

2018 ◽  
Vol 26 (7) ◽  
pp. 988-996
Author(s):  
Bryan F. Mitchell ◽  
Mei Chi ◽  
Elle Surgent ◽  
Bailey M. Sorochan ◽  
Curtis N. Tracey ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Ex Vivo ◽  
Neonatal Morbidity ◽  
The Novel ◽  
Uterine Contractility ◽  
Uterine Activity ◽  
Kinase C

Background: Preterm birth is the most common cause of neonatal morbidity and mortality and a common precedent to lifelong disability. Current treatment has minimal efficacy. Objective: We assessed the role of isozymes of the protein kinase C (PKC) family in regulating the phosphorylation of myosin regulatory light chains (RLCs), which regulate uterine contractility. We also explored the mechanisms through which these isozymes function. Study Design: We used a previously characterized and validated quantitative in-cell Western (ICW) assay to measure site-specific phosphorylations on myosin RLC and CPI-17. Cultures of human uterine myocytes (hUM) were treated with the potent contractile stimulant oxytocin to induce uterine contractility or a pharmacological mimic of diacyl-glycerol to stimulate the conventional and novel isozymes of the PKC family. Combinations of isozyme-selective inhibitors were used to determine the effects of the conventional and novel classes of isozymes. Results: Stimulation of PKC using phospho-dibutyrate caused immediate, concentration-dependent inhibition of uterine activity ex vivo. Using the ICW assay with hUM, the oxytocin-stimulated increase in the pro-contractile phosphorylations of myosin RLCs at serine19 and threonine18 was completely inhibited by prior treatment with phorbol-12-myristate-13-acetate, which stimulates both convention and novel classes of isozymes. Our results suggest that the conventional class of isozymes cause a reduction in phosphorylations at serine19 and threonine18 by reducing activity of myosin light chain kinase. The novel class of isozymes has 2 mechanisms of action: the first is activation of CPI-17 through phosphorylation at threonine38, which results in reduced activity of myosin light chain phosphatase and increased levels of activated myosin RLC; the second is increased phosphorylation of the N-terminal region of myosin RLC. Conclusions: Specific agonists for the conventional isozymes or inhibitors of the novel isozymes of the PKC family could be useful pharmacological agents for regulation of uterine activity.

scholarly journals The activation-induced decrease in the platelet surface expression of the glycoprotein Ib-IX complex is reversible

Blood ◽  
1994 ◽  
Vol 83 (12) ◽  
pp. 3562-3573 ◽  
Author(s):  
AD Michelson ◽  
SE Benoit ◽  
MH Kroll ◽  
JM Li ◽  
MJ Rohrer ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Surface Expression ◽  
Platelet Surface ◽  
Kinase C ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Thrombin decreases the platelet surface expression of the glycoprotein (GP) Ib-IX complex. To determine whether this effect is reversible, flow cytometric studies were performed with GPIb-IX-specific monoclonal antibodies. In both whole blood and washed platelet systems, incubation of platelets with thrombin or a combination of adenosine diphosphate and epinephrine resulted in a maximal decrease of the platelet surface expression of GPIb-IX within 5 minutes, after which there was a time- dependent return of the platelet surface GPIb-IX complex, which was maximal by 60 minutes. Exposure of the same platelets to additional exogenous thrombin resulted in a second decrease in platelet surface GPIb-IX, followed by a second reconstitution of platelet surface GPIb- IX. Throughout these experiments there was no measurable release from the platelets of glycocalicin (a proteolytic fragment of GPIb). Experiments in which platelets were preincubated with a biotinylated GPIb-specific MoAb showed that the GPIb molecules that returned to the platelet surface were the same molecules that had been translocated to the intraplatelet pool. The GPIb molecules that returned to the platelet surface were functionally competent to bind von Willebrand factor, as determined by ristocetin-induced platelet agglutination and ristocetin-induced binding of exogenous von Willebrand factor. Inhibitors of protein kinase C and myosin light-chain kinase enhanced the reexpression of platelet surface GPIb. In summary, the activation- induced decrease in the platelet surface expression of the GPIb-IX complex is reversible. Inactivation of protein kinase C and myosin light-chain kinase are important mechanisms in the reexpression of the platelet surface GPIb-IX complex.

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