Characteristics of the Interaction between Thrombin Exosite1 and the Sequence 269-297 of Platelet Glycoprotein Ibα

SummaryThe interaction between GPIb and thrombin promotes platelet activation elicited via the hydrolysis of the thrombin receptor and involves structures located on the segment 238-290 within the N-terminal domain of GPIbα and the positively charged exosite 1 on thrombin. We have investigated the ability of peptides derived from the 269-287 sequence of GPIbα to interact with thrombin. Three peptides were synthesized, including Ibα 269-287 and two scrambled peptides R1 and R2 which are comparable to Ibα 269-287 with regards to their content and distribution of anionic residues. However, R2 differs from both Ibα 269-287 and R1 by the shifting of one proline from a central position to the N-terminus. By chemical cross-linking, we observed the formation of a complex between 125I-Ibα 269-287 and α-thrombin that was inhibited by hirudin, the C-terminal peptide of hirudin, sodium pyrophosphate but not by heparin. The complex did not form when γ-thrombin was substituted for α-thrombin. Ibα 269-287 produced only slight changes in thrombin amidolytic activity and inhibited thrombin binding to fibrin. R1 and R2 also formed complexes with α-thrombin, modified slightly its catalytic activity and inhibited its binding to fibrin. Peptides Ibα 269-287 and R1 inhibited platelet aggregation and secretion induced by low thrombin concentrations whereas R2 was without effect. Our results indicate that Ibα 269-287 interacts with thrombin exosite 1 via mainly electrostatic interactions, which explains why the scrambled peptides also interact with exosite 1. Nevertheless, the lack of effect of R2 on thrombin-induced platelet activation suggests that proline 280 is important for thrombin interaction with GPIb.

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The C-terminal peptide of CCL21 drastically augments CCL21 activity through the dendritic cell lymph node homing receptor CCR7 by interaction with the receptor N-terminus

Cellular and Molecular Life Sciences ◽

10.1007/s00018-021-03930-7 ◽

2021 ◽

Author(s):

Astrid Sissel Jørgensen ◽

Emma Probst Brandum ◽

Jeppe Malthe Mikkelsen ◽

Klaudia A. Orfin ◽

Ditte Rahbæk Boilesen ◽

...

Keyword(s):

Lymph Node ◽

Electrostatic Interactions ◽

Immune Cell ◽

Direct Interaction ◽

Receptor Interaction ◽

Binding Model ◽

Lymph Node Homing ◽

Gating Mechanism ◽

N Terminus ◽

Positively Charged

AbstractThe endogenous chemokines CCL19 and CCL21 signal via their common receptor CCR7. CCL21 is the main lymph node homing chemokine, but a weak chemo-attractant compared to CCL19. Here we show that the 41-amino acid positively charged peptide, released through C-terminal cleavage of CCL21, C21TP, boosts the immune cell recruiting activity of CCL21 by up to 25-fold and the signaling activity via CCR7 by ~ 100-fold. Such boosting is unprecedented. Despite the presence of multiple basic glycosaminoglycan (GAG) binding motifs, C21TP boosting of CCL21 signaling does not involve interference with GAG mediated cell-surface retention. Instead, boosting is directly dependent on O-glycosylations in the CCR7 N-terminus. As dictated by the two-step binding model, the initial chemokine binding involves interaction of the chemokine fold with the receptor N-terminus, followed by insertion of the chemokine N-terminus deep into the receptor binding pocket. Our data suggest that apart from a role in initial chemokine binding, the receptor N-terminus also partakes in a gating mechanism, which could give rise to a reduced ligand activity, presumably through affecting the ligand positioning. Based on experiments that support a direct interaction of C21TP with the glycosylated CCR7 N-terminus, we propose that electrostatic interactions between the positively charged peptide and sialylated O-glycans in CCR7 N-terminus may create a more accessible version of the receptor and thus guide chemokine docking to generate a more favorable chemokine-receptor interaction, giving rise to the peptide boosting effect.

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Removing the Polyanionic Cargo Requirement for Assembly of Alphavirus Core-Like Particles to Make an Empty Alphavirus Core

Viruses ◽

10.3390/v12080846 ◽

2020 ◽

Vol 12 (8) ◽

pp. 846

Author(s):

Julie M. Button ◽

Suchetana Mukhopadhyay

Keyword(s):

Nucleic Acid ◽

Capsid Protein ◽

Electrostatic Interactions ◽

Wild Type ◽

N Terminus ◽

Charged Residues ◽

Size And Morphology ◽

Positively Charged

The assembly of alphavirus nucleocapsid cores requires electrostatic interactions between the positively charged N-terminus of the capsid protein (CP) and the encapsidated polyanionic cargo. This system differs from many other viruses that can self-assemble particles in the absence of cargo, or form “empty” particles. We hypothesized that the introduction of a mutant, anionic CP could replace the need for charged cargo during assembly. In this work, we produced a CP mutant, Minus 38 (M38), where all N-terminal charged residues are negatively-charged. When wild-type (WT) and M38 CPs were mixed, they assembled into core-like particles (CLPs). These “empty” particles were of similar size and morphology to WT CLPs assembled with DNA cargo, but did not contain nucleic acid. When DNA cargo was added to the assembly mixture, the amount of M38 CP that was assembled into CLPs decreased, but was not fully excluded from the CLPs, suggesting that M38 competes with DNA to interact with WT CPs. The composition of CLPs can be tuned by altering the order of addition of M38 CP, WT CP, and DNA cargo. The ability to produce alphavirus CLPs that contain a range of amounts of encapsidated cargo, including none, introduces a new platform for packaging cargo for delivery or imaging purposes.

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Reactivity in Micelles - Are We Really Able to Design Micellar Catalysts?

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc20080127 ◽

2008 ◽

Vol 73 (2) ◽

pp. 127-146 ◽

Cited By ~ 5

Author(s):

Radek Jurok ◽

Eva Svobodová ◽

Radek Cibulka ◽

František Hampl

Keyword(s):

Electrostatic Interactions ◽

Sodium Dodecyl ◽

Kinetic Studies ◽

Opposite Polarity ◽

Micellar Systems ◽

Ionic Micelles ◽

Diphenyl Phosphate ◽

Triton X ◽

Hydrolysis Of ◽

Positively Charged

Coordination of lipophilic alkyl pyridin-2-yl ketoximes 1 to Ni2+ ions, reduction of lipophilic 3-alkoxyacetophenones 2 with sodium borohydride, and alkaline hydrolysis of 4-nitrophenyl diphenyl phosphate (PNPDPP) were employed as probes in the investigation which factors may influence the reactivity of organic compounds in micellar systems. In all these reactions, a lipophilic substrate solubilized in micellar core was attacked by a hydrophilic reagent from the bulk aqueous phase. To evaluate the contribution of electrostatic interactions between the micellar surface charge and the reagent to the observed reactivity, we combined reactions involving the reagents with opposite polarity (Ni2+ cations and borohydride or hydroxide anions) with positively charged micelles of hexadecyltrimethylammonium chloride (CTAC) or bromide (CTAB) and negatively charged micelles of sodium dodecyl sulfate (SDS). Non-ionic micelles (Triton X-100 or Brij 35) served as a reference. The results of the kinetic studies give evidence that each of the investigated systems has unique properties going in particular aspects beyond the scope of the generally accepted concepts of reactivity in micelles.

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Platelet Activation and Aggregation Induced by Recombinant von Willebrand Factors Reproducing Four Type 2B von Willebrand Disease Missense Mutations

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614241 ◽

1998 ◽

Vol 79 (01) ◽

pp. 211-216 ◽

Cited By ~ 13

Author(s):

Lysiane Hilbert ◽

Claudine Mazurier ◽

Christophe de Romeuf

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Von Willebrand Disease ◽

Platelet Glycoprotein ◽

Missense Mutations ◽

Inhibit Platelet Aggregation ◽

Wild Type ◽

A1 Domain ◽

Von Willebrand ◽

Willebrand Factor

SummaryType 2B of von Willebrand disease (vWD) refers to qualitative variants with increased affinity of von Willebrand factor (vWF) for platelet glycoprotein Ib (GPIb). All the mutations responsible for type 2B vWD have been located in the A1 domain of vWF. In this study, various recombinant von Willebrand factors (rvWF) reproducing four type 2B vWD missense mutations were compared to wild-type rvWF (WT-rvWF) for their spontaneous binding to platelets and their capacity to induce platelet activation and aggregation. Our data show that the multimeric pattern of each mutated rvWF is similar to that of WT-rvWF but the extent of spontaneous binding and the capacity to induce platelet activation and aggregation are more important for the R543Q and V553M mutations than for the L697V and A698V mutations. Both the binding of mutated rvWFs to platelets and platelet aggregation induced by type 2B rvWFs are inhibited by monoclonal anti-GPIb and anti-vWF antibodies, inhibitors of vWF binding to platelets in the presence of ristocetin, as well as by aurin tricarboxylic acid. On the other hand, EDTA and a monoclonal antibody directed against GPIIb/IIIa only inhibit platelet aggregation. Furthermore, the incubation of type 2B rvWFs with platelets, under stirring conditions, results in the decrease in high molecular weight vWF multimers in solution, the extent of which appears correlated with that of plasma vWF from type 2B vWD patients harboring the corresponding missense mutation. This study supports that the binding of different mutated type 2B vWFs onto platelet GPIb induces various degrees of platelet activation and aggregation and thus suggests that the phenotypic heterogeneity of type 2B vWD may be related to the nature and/or location of the causative point mutation.

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Platelet activation by charged ligands and nanoparticles: platelet glycoprotein receptors as pattern recognition receptors

Platelets ◽

10.1080/09537104.2021.1945571 ◽

2021 ◽

pp. 1-13

Author(s):

Samantha J. Montague ◽

Pushpa Patel ◽

Eleyna M. Martin ◽

Alexandre Slater ◽

Lourdes Garcia Quintanilla ◽

...

Keyword(s):

Pattern Recognition ◽

Platelet Activation ◽

Pattern Recognition Receptors ◽

Platelet Glycoprotein ◽

Charged Ligands

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How Insertion of a Single Tryptophan in the N-Terminus of a Cecropin A-Melittin Hybrid Peptide Changes Its Antimicrobial and Biophysical Profile

Membranes ◽

10.3390/membranes11010048 ◽

2021 ◽

Vol 11 (1) ◽

pp. 48

Author(s):

Ana Rita Ferreira ◽

Cátia Teixeira ◽

Carla F. Sousa ◽

Lucinda J. Bessa ◽

Paula Gomes ◽

...

Keyword(s):

Electrostatic Interactions ◽

Bacterial Infections ◽

Electron System ◽

Bacterial Membranes ◽

Highly Active ◽

Large Unilamellar Vesicles ◽

Biophysical Profile ◽

N Terminus ◽

Lysine Residues ◽

Preferential Location

In the era of antibiotic resistance, there is an urgent need for efficient antibiotic therapies to fight bacterial infections. Cationic antimicrobial peptides (CAMP) are promising lead compounds given their membrane-targeted mechanism of action, and high affinity towards the anionic composition of bacterial membranes. We present a new CAMP, W-BP100, derived from the highly active BP100, holding an additional tryptophan at the N-terminus. W-BP100 showed a broader antibacterial activity, demonstrating a potent activity against Gram-positive strains. Revealing a high partition constant towards anionic over zwitterionic large unilamellar vesicles and inducing membrane saturation at a high peptide/lipid ratio, W-BP100 has a preferential location for hydrophobic environments. Contrary to BP100, almost no aggregation of anionic vesicles is observed around saturation conditions and at higher concentrations no aggregation is observed. With these results, it is possible to state that with the incorporation of a single tryptophan to the N-terminus, a highly active peptide was obtained due to the π–electron system of tryptophan, resulting in negatively charged clouds, that participate in cation–π interactions with lysine residues. Furthermore, we propose that W-BP100 action can be achieved by electrostatic interactions followed by peptide translocation.

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Clusters of Charged Residues at the C Terminus of MotA and N Terminus of MotB Are Important for Function of the Escherichia coli Flagellar Motor

Journal of Bacteriology ◽

10.1128/jb.00407-08 ◽

2008 ◽

Vol 190 (15) ◽

pp. 5517-5521 ◽

Cited By ~ 11

Author(s):

Edan R. Hosking ◽

Michael D. Manson

Keyword(s):

Escherichia Coli ◽

Flagellar Motor ◽

Protein Levels ◽

Partner Protein ◽

C Terminus ◽

N Terminus ◽

Charged Residues ◽

Positively Charged ◽

Terminal Cluster

ABSTRACT MotA contains a conserved C-terminal cluster of negatively charged residues, and MotB contains a conserved N-terminal cluster of positively charged residues. Charge-altering mutations affecting these residues impair motility but do not diminish Mot protein levels. The motility defects are reversed by second-site mutations targeting the same or partner protein.

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Abstract 11947: Unraveling the Mechanism for the Stimulatory Role of Platelet GPIb-IX-V in Thrombin-Induced Platelet Activation

Circulation ◽

10.1161/circ.130.suppl_2.11947 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Brian Estevez ◽

Michael K Delaney ◽

Aleksandra Stojanovic-Terpo ◽

Xiaoping Du

Keyword(s):

Platelet Activation ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Human Platelets ◽

Platelet Glycoprotein ◽

Inhibitory Peptide ◽

Protease Activated Receptors ◽

Signaling Mechanism ◽

Ovary Cells

Numerous reports indicate that the platelet glycoprotein (GP) Ib-IX complex (GPIb-IX) binds directly to the potent platelet agonist thrombin and is important for promoting thrombin-induced platelet activation. However, how GPIb-IX contributes to thrombin-induced platelet activation is unclear. It has been suggested that thrombin binding to GPIb facilitates the cleavage, and thus activation, of the protease-activated receptors (PAR). Our data indicate that GPIb-IX promotes thrombin signaling through a GPIb-IX signaling mechanism. Pretreatment of human platelets with MPalphaC, an inhibitory peptide based on a critical 14-3-3 signaling protein binding site on the cytoplasmic domain of the GPIb alpha chain, inhibited thrombin-induced platelet activation. MPalphaC-treatment inhibited thrombin-induced activation of Rac1 and LIMK1, both of which are known to play essential roles in GPIb signaling. To more specifically determine the role of GPIb-IX, we reconstituted GPIb-IX-facilitated thrombin signaling in Chinese Hamster Ovary cells expressing PAR1. Thrombin induced signaling was significantly enhanced by GPIb-expression, and deletion of the cytoplasmic 14-3-3-binding domain of GPIb alpha abolished the stimulatory effect of GPIb on thrombin signaling. Furthermore, the role of GPIb-IX in promoting thrombin signaling requires Rac1, and GPIb-IX-dependent Rac1 activation and LIMK phosphorylation are abolished in delta 605 cells expressing a 14-3-3-binding defective mutant GPIb alpha. Taken together, these data suggest that the stimulatory role of GPIb in thrombin signaling requires a C-terminal 14-3-3-binding region which mediates activation of a Rac1/LIMK1 pathway that promotes thrombin signaling leading to platelet activation.

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Aprotinin: is it prothrombotic?

Perfusion ◽

10.1177/026765910101600510 ◽

2001 ◽

Vol 16 (5) ◽

pp. 401-409 ◽

Cited By ~ 3

Author(s):

M Poullis ◽

R C Landis ◽

K M Taylor

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Mechanism Of Action ◽

Adenosine Diphosphate ◽

Platelet Membrane ◽

Calcium Flux ◽

Thrombin Receptor ◽

Proteolytic Activation ◽

Agonist Peptide ◽

Receptor Family

Controversy continues as to whether aprotinin (Trasylol) is prothrombotic. The recent discovery of the thrombin receptor family, known as the protease-activated receptor family (PAR) has been essential in aiding our understanding of the mechanism of action of aprotinin. Our results show that aprotinin has no effect on platelet aggregation induced by adrenaline, adenosine diphosphate, phorbol-12-myristate-13-acetate, collagen or PAR 1 agonist peptide. However, aprotinin inhibits thrombin-induced platelet activation as assessed by macroaggregation, microaggregation and platelet membrane calcium flux. Aprotinin inhibits proteolytic activation of platelets, but platelets can still be activated by non-proteolytic mechanisms.

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Role of a tryptophan anchor in human topoisomerase I structure, function and inhibition

Biochemical Journal ◽

10.1042/bj20071436 ◽

2008 ◽

Vol 411 (3) ◽

pp. 523-530 ◽

Cited By ~ 12

Author(s):

Gary S. Laco ◽

Yves Pommier

Keyword(s):

Amino Acids ◽

Topoisomerase I ◽

Electrostatic Interactions ◽

Functional Domain ◽

Site Mutation ◽

Supercoiled Dna ◽

Terminal Domain ◽

Tryptophan Residues ◽

N Terminus

Human Top1 (topoisomerase I) relaxes supercoiled DNA during cell division and transcription. Top1 is composed of 765 amino acids and contains an unstructured N-terminal domain of 200 amino acids, and a structured functional domain of 565 amino acids that binds and relaxes supercoiled DNA. In the present study we examined the region spanning the junction of the N-terminal domain and functional domain (junction region). Analysis of several published Top1 structures revealed that three tryptophan residues formed a network of aromatic stacking interactions and electrostatic interactions that anchored the N-terminus of the functional domain to sub-domains containing the nose cone and active site. Mutation of the three tryptophan residues (Trp203/Trp205/Trp206) to an alanine residue, either individually or together, in silico revealed that the individual tryptophan residue's contribution to the tryptophan ‘anchor’ was additive. When the three tryptophan residues were mutated to alanine in vitro, the resulting mutant Top1 differed from wild-type Top1 in that it lacked processivity, exhibited resistance to camptothecin and was inactivated by urea. The results indicated that the tryptophan anchor stabilized the N-terminus of the functional domain and prevented the loss of Top1 structure and function.

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