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.

Phosphorylation Of Platelet Actin-Binding Protein During Platelet Activation

1981 ◽  
Author(s):  
Roger C Carroll ◽  
Jonathan M Gerrard
Keyword(s):  
Molecular Weight ◽  
Platelet Activation ◽  
Time Course ◽  
Binding Protein ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Actin Binding ◽  
Actin Binding Protein ◽  
Platelet Protein ◽  
External Calcium

We have followed the 32P-labelling of actin-binding protein as a function of platelet activation. Utilizing polyacrylamide sodium dodecyl sulfate gel electrophoresis to resolve total platelet protein samples we found 2 to 3 fold labelling increases in actin-binding protein 30 to 60 seconds after thrombin stimulation. Somewhat larger increases were observed for 40,000 and 20,000 apparent molecular weight peptides. The actin-binding protein was identified on the gels by coelectrophoresis of purified actin-binding protein as well as cytoskeletal cores prepared by detergent extraction of activated 32p-iabelled platelets. In addition, these cytoskeletal cores indicated that the 32P-labelled actin-binding protein was closely associated with the activated platelet's cytoskeleton. Following the 32P-labelling of actin-binding protein over an 8 minute time course revealed that in aggregating platelet samples rapid desphosphorylation to almost initial levels occurred between 3 and 5 minutes. A similar curve was obtained for the 20,000 apparent molecular weight peptide. This rapid dephosphorylation was shown to be dependent on platelet aggregation in the absence of external calcium or in thrombastenic platelets lacking the aggregation response to activation. These results suggest that phosphorylation of actin-binding protein initiates its association with the platelet cytoskeleton during activation.

scholarly journals Phosphorylation of platelet actin-binding protein during platelet activation

Blood ◽  
1982 ◽  
Vol 59 (3) ◽  
pp. 466-471 ◽  
Author(s):  
RC Carroll ◽  
JM Gerrard
Keyword(s):  
Molecular Weight ◽  
Platelet Activation ◽  
Time Course ◽  
Binding Protein ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Actin Binding ◽  
Cell Contact ◽  
Actin Binding Protein ◽  
Platelet Protein

Abstract In this study we have followed the 32P-labeling of actin-binding protein as a function of platelet activation. Utilizing polyacrylamide- sodium dodecyl sulfate gel electrophoresis to resolve total platelet protein samples, we found 2--3-fold labeling increases in actin-binding protein 30--60 sec after thrombin stimulation. Somewhat larger increases were observed for 40,000 and 20,000 apparent molecular weight peptides. The actin-binding protein was identified on the gels by coelectrophoresis with purified actin-binding protein, its presence in cytoskeletal cores prepared by detergent extraction of activated 32P- labeled platelets, and by direct immunoprecipitation with antibodies against guinea pig vas deferens filamin (actin-binding protein). In addition, these cytoskeletal cores indicated that the 32P-labeled actin- binding protein was closely associated with the activated platelet's cytoskeleton. Following the 32P-labeling of actin-binding protein over an 8-min time course revealed that in aggregating platelet samples rapid dephosphorylation to almost initial levels occurred between 3 and 5 min. A similar curve was obtained for the 20,000 apparent molecular weight peptide. However, rapid dephosphorylation was not observed if platelet aggregation was prevented by chelating external calcium or by using thrombasthenic platelets lacking the aggregation response. Thus, cell-cell contact would seem to be crucial in initiating the rapid dephosphorylation response.

scholarly journals Calponin, an actin-binding protein, concerns platelet activation

2000 ◽  
Vol 82 ◽  
pp. 54
Author(s):  
Kazunao Kondo ◽  
Takashi Tsuboi ◽  
Susumu Terakawa ◽  
Katsuhito Takahash ◽  
Yasuhiro Suzuki ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Binding Protein ◽  
Actin Binding ◽  
Actin Binding Protein

scholarly journals Phosphorylation of platelet actin-binding protein during platelet activation

Blood ◽  
1982 ◽  
Vol 59 (3) ◽  
pp. 466-471 ◽  
Author(s):  
RC Carroll ◽  
JM Gerrard
Keyword(s):  
Molecular Weight ◽  
Platelet Activation ◽  
Time Course ◽  
Binding Protein ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Actin Binding ◽  
Cell Contact ◽  
Actin Binding Protein ◽  
Platelet Protein

In this study we have followed the 32P-labeling of actin-binding protein as a function of platelet activation. Utilizing polyacrylamide- sodium dodecyl sulfate gel electrophoresis to resolve total platelet protein samples, we found 2--3-fold labeling increases in actin-binding protein 30--60 sec after thrombin stimulation. Somewhat larger increases were observed for 40,000 and 20,000 apparent molecular weight peptides. The actin-binding protein was identified on the gels by coelectrophoresis with purified actin-binding protein, its presence in cytoskeletal cores prepared by detergent extraction of activated 32P- labeled platelets, and by direct immunoprecipitation with antibodies against guinea pig vas deferens filamin (actin-binding protein). In addition, these cytoskeletal cores indicated that the 32P-labeled actin- binding protein was closely associated with the activated platelet's cytoskeleton. Following the 32P-labeling of actin-binding protein over an 8-min time course revealed that in aggregating platelet samples rapid dephosphorylation to almost initial levels occurred between 3 and 5 min. A similar curve was obtained for the 20,000 apparent molecular weight peptide. However, rapid dephosphorylation was not observed if platelet aggregation was prevented by chelating external calcium or by using thrombasthenic platelets lacking the aggregation response. Thus, cell-cell contact would seem to be crucial in initiating the rapid dephosphorylation response.

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.

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.

Clathrin Hub Expression Dissociates the Actin-Binding Protein Hip1R from Coated Pits and Disrupts Their Alignment with the Actin Cytoskeleton

Traffic ◽  
2001 ◽  
Vol 2 (11) ◽  
pp. 851-858 ◽  
Author(s):  
Elizabeth M. Bennett ◽  
Chih-Ying Chen ◽  
Asa E. Y. Engqvist-Goldstein ◽  
David G. Drubin ◽  
Frances M. Brodsky
Keyword(s):  
Actin Cytoskeleton ◽  
Binding Protein ◽  
Actin Binding ◽  
Coated Pits ◽  
Actin Binding Protein

Binding of Human Platelet Glycoprotein lb and Actin to Fragments of Actin-Binding Protein

1992 ◽  
Vol 67 (02) ◽  
pp. 252-257 ◽  
Author(s):  
Anne M Aakhus ◽  
J Michael Wilkinson ◽  
Nils Olav Solum
Keyword(s):  
Actin Filaments ◽  
High Speed ◽  
Binding Protein ◽  
Protein A ◽  
Actin Binding ◽  
Platelet Glycoprotein ◽  
Actin Binding Protein ◽  
Crossed Immunoelectrophoresis ◽  
Triton X ◽  
Calpain Inhibitors

SummaryActin-binding protein (ABP) is degraded into fragments of 190 and 90 kDa by calpain. A monoclonal antibody (MAb TI10) against the 90 kDa fragment of ABP coprecipitated with the glycoprotein lb (GP lb) peak observed on crossed immunoelectrophoresis of Triton X-100 extracts of platelets prepared without calpain inhibitors. MAb PM6/317 against the 190 kDa fragment was not coprecipitated with the GP lb peak under such conditions. The 90 kDa fragment was adsorbed on protein A agarose from extracts that had been preincubated with antibodies to GP lb. This supports the idea that the GP Ib-ABP interaction resides in the 90 kDa region of ABP. GP lb was sedimented with the Triton-insoluble actin filaments in trace amounts only, and only after high speed centrifugation (100,000 × g, 3 h). Both the 190 kDa and the 90 kDa fragments of ABP were sedimented with the Triton-insoluble actin filaments.

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