Abstract 212: Thrombin Stimulation Induces de novo SDF-1a Synthesis and Alters MicroRNA Profile in Human Platelets

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Zhangsen Huang ◽  
Mohammed Ferdous-Ur Rahman ◽  
Lei Jiang ◽  
Hong Xie ◽  
Stefano Caramuta ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Platelet Activation ◽  
Mirna Expression ◽  
De Novo ◽  
Platelet Rich Plasma ◽  
Mrna Translation ◽  
Human Platelets ◽  
Mirna Array ◽  
Mirna Expression Profiling ◽  
De Novo Protein Synthesis

Platelets are anucleated cells, but still retain certain capacity of protein synthesis. Platelets are also known to contain microRNAs (miRNAs), which can negatively regulate mRNA translation/protein synthesis. We asked if platelet activation can initiate de novo protein synthesis of angiogenic regulators and if platelet activation induces changes of platelet miRNA expression. Platelet rich plasma was prepared from citrated blood of 12 healthy subjects. Platelets were purified by a second centrifugation and using leukocyte-depleting CD45-Dynabeads, and then treated without or with thrombin (0.1 U/ml, 30 min, 37°C). Platelet lysates were prepared immediately after treatments or after 16 h-culture for Western blottings of stromal cell derived factor-1a (SDF1a) and angiostatin. Total platelet RNA was isolated with a mirVana miRNA isolation kit, and platelet miRNA expression profiling was performed using an Agilent’s miRNA microarray system. Thrombin stimulation reduced platelet immunoblotting intensities/protein contents of SDF-1a and angiostatin as compared to unstimulated platelets, suggesting release of the angiogenic regulators upon platelet activation. SDF-1a, but not angiostatin, content was partially recovered after 16 h culture, indicating de novo protein synthesis. Using mature mRNA-specific primers, SDF-1a, but not angiostatin, mRNA was detected in thrombin-activated but not in unstimulated platelets. Furthermore, miRNA array analysis demonstrated that thrombin stimulation down-regulated a panel of platelet miRNA expression (e.g., miR-23a, miR-23b, miR-24, miR-106b, and miR-107) but also up-regulated a group of miRNA expression (e.g., miR-96, miR-212, miR-449a, and miR-629). In conclusion, thrombin stimulation induces mRNA splicing and protein synthesis of SDF-1a in platelets. Thrombin activation also alters platelet miRNA profile that may have a major impact on de novo protein synthesis in activated platelets.

Abstract 17411: De Novo Protein Synthesis of Alpha-Toxin Activated Platelets

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Michael Buerke ◽  
Linda Bingener ◽  
Priyanka Boettger
Keyword(s):  
Staphylococcus Aureus ◽  
Protein Synthesis ◽  
Platelet Activation ◽  
De Novo ◽  
Binding Protein ◽  
Actin Binding ◽  
High Morbidity ◽  
Actin Binding Protein ◽  
De Novo Protein Synthesis ◽  
Phosphorylation Analysis

Introduction: Endovascular infections with bacteria are often devastating with subsequent high morbidity and mortality. Exo- and or endotoxins of bacteria can activate endothelial cells, leukocytes and platelets. Platelets are first line defence they accumulate at sites of vascular injury or infection. Platelet activation is a necessary step in thrombus formation. Nevertheless, stimulation of platelets will result in de novo protein synthesis despite missing nucleus since platelets armed with translational equipment. Methods: In the present study we determined the effect of staphylococcus aureus α-toxin on platelet activation and de novo protein synthesis analysed with 2-D gels, proteomics and phosphorylation analysis. Results: α-toxin induced platelet activation resulted in modulation of de novo protein synthesis of DJ-1 Protein, ras suppressor protein1, PLEK protein, fumaryl aceto acetase sowie das coronin actin binding protein. This synthesis was time- and concentration-dependent and was markedly increased when platelets adhered to collagen or fibrinogen and required ligation of α IIb β 3 . Accumulation of protein synthesis in platelets was blocked by global translational inhibitors and attenuated by inhibitors that regulate signalling through the mammalian Target of Rapamycin (mTOR). In addition with phosphorylation analysis we were able demonstrate modulation of threonine phosphorylation of fumaryl aceto acetase, phosphor threonin signal of coronin actin binding protein, phosphorylation of peroxiredoxin-6, phosphorylation of tropomyosin-2, phosphothreonin signal of H + transporting two sector ATPase upon α-toxin stimulation. Conclusion: Interactions with staphylococcus aureus α-toxin and platelets might lead to their activation and de novo protein synthesis. These results suggest that platelets have an important role in inflammation besides their aggregating duties in inflammatory disease.

Amino Acid Transport into Human Platelets and Subsequent Incorporation into Protein

1970 ◽  
Vol 23 (01) ◽  
pp. 140-147 ◽  
Author(s):  
I. A Cooper ◽  
B. G Firkin
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Amino Acid Transport ◽  
De Novo ◽  
Human Platelets ◽  
Acid Transport ◽  
Protein Components ◽  
De Novo Protein Synthesis ◽  
Subsequent Incorporation

SummaryHuman platelets are known to contain various protein components. Among them are fibrinogen and other soluble proteins. The origin of such proteins has not been clear.Studies were designed to demonstrate the ability of the platelet to incorporate amino acids and subsequently utilise these for de novo protein synthesis.Seven different 14C labelled amino acids were used, cysteine, lysine, serine, glutamic acid, proline, valine und leucine.Active incorporation into platelets of all these amino acids was demonstrated with some evidence for incorporation of cysteine, lysine and serine into the fibrinogen and soluble protein.

Dissociation of SERPINE1 mRNA from the translational repressor proteins Ago2 and TIA-1 upon platelet activation

2015 ◽  
Vol 113 (05) ◽  
pp. 1046-1059 ◽  
Author(s):  
Aurélie Corduan ◽  
Hélène Plé ◽  
Benoit Laffont ◽  
Thérèse Wallon ◽  
Isabelle Plante ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Platelet Activation ◽  
De Novo ◽  
Protein Complexes ◽  
Translational Repression ◽  
Messenger Rnas ◽  
Plasminogen Activator Inhibitor 1 ◽  
Translational Repressor ◽  
De Novo Protein Synthesis ◽  
Pai 1

SummaryPlatelets play an important role in haemostasis, as well as in thrombosis and coagulation processes. They harbour a wide variety of messenger RNAs (mRNAs), that can template de novo protein synthesis, and an abundant array of microRNAs, which are known to mediate mRNA translational repression through proteins of the Argonaute (Ago) family. The relationship between platelet microRNAs and proteins capable of mediating translational repression, however, remains unclear. Here, we report that half of platelet microRNAs is associated to mRNA-regulatory Ago2 protein complexes, in various proportions. Associated to these Ago2 complexes are platelet mRNAs known to support de novo protein synthesis. Reporter gene activity assays confirmed the capacity of the platelet microRNAs, found to be associated to Ago2 complexes, to regulate translation of these platelet mRNAs through their 3’UTR. Neither the microRNA repertoire nor the microRNA composition of Ago2 complexes of human platelets changed upon activation with thrombin. However, under conditions favoring de novo synthesis of Plasminogen Activator Inhibitor-1 (PAI-1) protein, we documented a rapid dissociation of the encoding platelet SERPINE1 mRNA from Ago2 protein complexes as well as from the translational repressor protein T-cell-restricted intracellular antigen-1 (TIA-1). These findings are consistent with a scenario by which lifting of the repressive effects of Ago2 and TIA-1 protein complexes, involving a rearrangement of protein•mRNA complexes rather than disassembly of Ago2•microRNA complexes, would allow translation of SERPINE1 mRNA into PAI-1 in response to platelet activation.

scholarly journals Viral Gene Expression Patterns in Human Herpesvirus 6B-Infected T Cells

2002 ◽  
Vol 76 (15) ◽  
pp. 7578-7586 ◽  
Author(s):  
Bodil Øster ◽  
Per Höllsberg
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Protein Synthesis ◽  
De Novo ◽  
Expression Patterns ◽  
Human Herpesvirus ◽  
Viral Gene Expression ◽  
Polymerase Activity ◽  
Viral Gene ◽  
De Novo Protein Synthesis

ABSTRACT Herpesvirus gene expression is divided into immediate-early (IE) or α genes, early (E) or β genes, and late (L) or γ genes on the basis of temporal expression and dependency on other gene products. By using real-time PCR, we have investigated the expression of 35 human herpesvirus 6B (HHV-6B) genes in T cells infected by strain PL-1. Kinetic analysis and dependency on de novo protein synthesis and viral DNA polymerase activity suggest that the HHV-6B genes segregate into six separate kinetic groups. The genes expressed early (groups I and II) and late (groups V and VI) corresponded well with IE and L genes, whereas the intermediate groups III and IV contained E and L genes. Although HHV-6B has characteristics similar to those of other roseoloviruses in its overall gene regulation, we detected three B-variant-specific IE genes. Moreover, genes that were independent of de novo protein synthesis clustered in an area of the viral genome that has the lowest identity to the HHV-6A variant. The organization of IE genes in an area of the genome that differs from that of HHV-6A underscores the distinct differences between HHV-6B and HHV-6A and may provide a basis for further molecular and immunological analyses to elucidate their different biological behaviors.

De novo protein synthesis in relation to ammonia and proline accumulation in water stressed white clover

2004 ◽  
Vol 31 (8) ◽  
pp. 847 ◽  
Author(s):  
Tae-Hwan Kim ◽  
Bok-Rye Lee ◽  
Woo-Jin Jung ◽  
Kil-Yong Kim ◽  
Jean-Christophe Avice ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Water Deficit ◽  
White Clover ◽  
De Novo ◽  
Proline Accumulation ◽  
Leaf Water ◽  
Total N ◽  
Water Parameters ◽  
Leaves And Roots ◽  
De Novo Protein Synthesis

The kinetics of protein incorporation from newly-absorbed nitrogen (N, de novo protein synthesis) was estimated by 15N tracing in 18-week-old white clover plants (Trifolium repens L. cv. Regal) during 7 d of water-deficit treatment. The physiological relationship between kinetics and accumulation of proline and ammonia in response to the change in leaf-water parameters was also assessed. All leaf-water parameters measured decreased gradually under water deficit. Leaf and root dry mass was not significantly affected during the first 3 d when decreases in leaf-water parameters were substantial. However, metabolic parameters such as total N, proline and ammonia were significantly affected within 1 d of commencement of water-deficit treatment. Water-deficit treatment significantly increased the proline and NH3–NH4+ concentrations in both leaves and roots. There was a marked reduction in the amount of N incorporated into the protein fraction from the newly absorbed N (NANP) in water-deficit stressed plants, particularly in leaf tissue. This reduction in NANP was strongly associated with an increased concentration of NH3–NH4+ in roots (P≤0.05) and proline (P≤0.01) in leaves and roots. These results suggest that proline accumulation may be a sensitive biochemical indicator of plant water status and of the dynamics of de novo protein synthesis in response to stress severity.

scholarly journals Activation of human platelets by immune complexes prepared with cationized human IgG

Blood ◽  
1993 ◽  
Vol 82 (10) ◽  
pp. 3045-3051
Author(s):  
M Schattner ◽  
M Lazzari ◽  
AS Trevani ◽  
E Malchiodi ◽  
AC Kempfer ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Immune Complexes ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Human Igg ◽  
Experimental Conditions ◽  
Induce Platelet Aggregation ◽  
Soluble Immune Complexes ◽  
High Concentrations

The present study shows that the ability of soluble immune complexes (IC), prepared with human IgG and rabbit IgG antibodies against human IgG, to trigger platelet activation was markedly higher for IC prepared with cationized human IgG (catIC) compared with those prepared with untreated human IgG (cIC). CatIC induced platelet aggregation and adenosine triphosphate release in washed platelets (WP), gel-filtered platelets (GFP), or platelet-rich plasma (PRP) at physiologic concentrations of platelets (3 x 10(8)/mL) and at low concentrations of catIC (1 to 30 micrograms/mL). On the contrary, under similar experimental conditions, cIC did not induce aggregation in PRP, WP, or GFP. Low aggregation responses were only observed using high concentrations of both WP (9 x 10(8)/mL) and cIC (500 micrograms/mL). Interestingly, catIC were also able to induce platelet activation under nonaggregating conditions, as evidenced by P-selectin expression. Cationized human IgG alone did not induce platelet aggregation in PRP but triggered either WP or GFP aggregation. However, the concentration needed to induce these responses, was about eightfold higher than those required for catIC. The responses induced either by catIC or cationized human IgG were completely inhibited by treatment with heparin, dextran sulphate, EDTA, prostaglandin E1, or IV3, a monoclonal antibody against the receptor II for the Fc portion of IgG (Fc gamma RII). The data presented in this study suggest that IgG charge constitutes a critical property that conditions the ability of IC to trigger platelet activation.

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