scholarly journals A Synthetic Triple Helical Collagen Peptide as a New Agonist for Flow Cytometric Measurement of GPVI-Specific Platelet Activation

2019 ◽  
Vol 119 (12) ◽  
pp. 2005-2013
Author(s):  
Yaqiu Sang ◽  
Dana Huskens ◽  
Kanin Wichapong ◽  
Bas de Laat ◽  
Gerry A. F. Nicolaes ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Platelet Function ◽  
Good Alternative ◽  
Interindividual Variation ◽  
Glycoprotein Vi ◽  
Collagen Peptide ◽  
C Terminus ◽  
Helical Peptide ◽  
N Terminus ◽  
Flow Cytometric Measurement

AbstractSynthetic cross-linked collagen-related peptide (CRP-XL) is a glycoprotein VI (GPVI) receptor activator for platelet activation. This triple helical peptide, widely used in platelet function tests, is synthesized and cross-linked through cysteine residues at its N-terminus and C-terminus. Currently, there is only one laboratory, which is capable to produce this valuable peptide for clinical applications. In an attempt to provide a standardized alternative for CRP-XL, we developed a synthetic triple helical collagen peptide (STH-CP) with the same primary sequence as CRP-XL (GPC-(GPO)10-GPCG-amide)3, which was both on the C-terminus and on the N-terminus fixed on a scaffold with a binding side for each of the three peptides. The performance of STH-CP on platelet function was studied using flow cytometry and compared with CRP-XL. We found that platelet activation pattern in response to STH-CP and CRP-XL is similar, although the STH-CP requires sixfold higher concentrations to activate platelets to the same state. The intra-assay percent coefficient of variation of STH-CP and CRP-XL were both < 5% and the interindividual variation measured in 118 individuals for both peptides was around 23 and 21% for αIIbβ3 activation and P-selectin expression, respectively. The STH-CP in ready-to-use reaction mix has lower variation than CRP-XL over 1-year storage. In reference values and seasonal variation study, the platelet activation response showed a strong correlation between STH-CP and CRP-XL.Our findings show that this new STH-CP is a stable and potent platelet GPVI agonist which can induce the same reproducible platelet activation as CRP-XL and that STH-CP can be considered as a good alternative for CRP-XL.

scholarly journals In-depth PtdIns(3,4,5)P3 signalosome analysis identifies DAPP1 as a negative regulator of GPVI-driven platelet function

2017 ◽  
Vol 1 (14) ◽  
pp. 918-932 ◽  
Author(s):  
Tom N. Durrant ◽  
James L. Hutchinson ◽  
Kate J. Heesom ◽  
Karen E. Anderson ◽  
Len R. Stephens ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Platelet Function ◽  
Binding Proteins ◽  
Thrombus Formation ◽  
Negative Regulator ◽  
Valuable Resource ◽  
Future Studies ◽  
Glycoprotein Vi ◽  
Key Points ◽  
Depth Analysis

Key Points We present the first in-depth analysis of platelet PtdIns(3,4,5)P3-binding proteins, providing a valuable resource for future studies. The PtdIns(3,4,5)P3-binding protein, DAPP1, negatively regulates glycoprotein VI–driven platelet activation and thrombus formation.

Better platelet function, less fibrinolysis and less hemolysis in re-transfused residual pump blood with the Ringer’s chase technique – a randomized pilot study

Perfusion ◽  
2017 ◽  
Vol 33 (3) ◽  
pp. 185-193 ◽  
Author(s):  
Anki Olsson ◽  
Joakim Alfredsson ◽  
Sofia Ramström ◽  
Rolf Svedjeholm ◽  
Dermot Kenny ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Platelet Function ◽  
Artery Bypass ◽  
Bypass Graft ◽  
Adenosine Diphosphate ◽  
Activation Markers ◽  
Glycoprotein Vi ◽  
Impedance Aggregometry ◽  
Residual Blood ◽  
And Function

Introduction: Residual pump blood from the cardiopulmonary bypass (CPB) circuit is often collected into an infusion bag (IB) and re-transfused. An alternative is to chase the residual blood into the circulation through the arterial cannula with Ringer’s acetate. Our aim was to assess possible differences in hemostatic blood quality between these two techniques. Methods: Forty adult patients undergoing elective coronary artery bypass graft surgery with CPB were randomized to receive the residual pump blood by either an IB or through the Ringer’s chase (RC) technique. Platelet activation and function (impedance aggregometry), coagulation and hemolysis variables were assessed in the re-transfused blood and in the patients before, during and after surgery. Results are presented as median (25-75 quartiles). Results: Total hemoglobin and platelet levels in the re-transfused blood were comparable with the two methods, as were soluble platelet activation markers P-selectin and soluble glycoprotein VI (GPVI). Platelet aggregation (U) in the IB blood was significantly lower compared to the RC blood, with the agonists adenosine diphosphate (ADP) 24 (10-32) vs 46 (33-65), p<0.01, thrombin receptor activating peptide (TRAP) 50 (29-73) vs 69 (51-92), p=0.04 and collagen 24 (17-28) vs 34 (26-59), p<0.01. The IB blood had higher amounts of free hemoglobin (mg/L) (1086 (891-1717) vs 591(517-646), p<0.01) and D-dimer 0.60 (0.33-0.98) vs 0.3 (0.3-0.48), p<0.01. Other coagulation variables showed no difference between the groups. Conclusions: The handling of blood after CPB increases hemolysis, impairs platelet function and activates coagulation and fibrinolysis. The RC technique preserved the blood better than the commonly used IB technique.

scholarly journals Oral administration of Bruton's tyrosine kinase inhibitors impairs GPVI-mediated platelet function

2016 ◽  
Vol 310 (5) ◽  
pp. C373-C380 ◽  
Author(s):  
Rachel A. Rigg ◽  
Joseph E. Aslan ◽  
Laura D. Healy ◽  
Michael Wallisch ◽  
Marisa L. D. Thierheimer ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Platelet Activation ◽  
Platelet Function ◽  
Lymphocytic Leukemia ◽  
Bruton’S Tyrosine Kinase ◽  
Bruton's Tyrosine Kinase ◽  
Glycoprotein Vi ◽  
Activation Spreading

The Tec family kinase Bruton's tyrosine kinase (Btk) plays an important signaling role downstream of immunoreceptor tyrosine-based activation motifs in hematopoietic cells. Mutations in Btk are involved in impaired B-cell maturation in X-linked agammaglobulinemia, and Btk has been investigated for its role in platelet activation via activation of the effector protein phospholipase Cγ2 downstream of the platelet membrane glycoprotein VI (GPVI). Because of its role in hematopoietic cell signaling, Btk has become a target in the treatment of chronic lymphocytic leukemia and mantle cell lymphoma; the covalent Btk inhibitor ibrutinib was recently approved by the US Food and Drug Administration for treatment of these conditions. Antihemostatic events have been reported in some patients taking ibrutinib, although the mechanism of these events remains unknown. We sought to determine the effects of Btk inhibition on platelet function in a series of in vitro studies of platelet activation, spreading, and aggregation. Our results show that irreversible inhibition of Btk with two ibrutinib analogs in vitro decreased human platelet activation, phosphorylation of Btk, P-selectin exposure, spreading on fibrinogen, and aggregation under shear flow conditions. Short-term studies of ibrutinib analogs administered in vivo also showed abrogation of platelet aggregation in vitro, but without measurable effects on plasma clotting times or on bleeding in vivo. Taken together, our results suggest that inhibition of Btk significantly decreased GPVI-mediated platelet activation, spreading, and aggregation in vitro; however, prolonged bleeding was not observed in a model of bleeding.

scholarly journals G protein-coupled receptor kinase 5 regulates thrombin signaling in platelets via PAR-1

2021 ◽  
Author(s):  
Kate Downes ◽  
Xuefei Zhao ◽  
Nicholas S Gleadall ◽  
Harriet McKinney ◽  
Carly Kempster ◽  
...  
Keyword(s):  
G Protein ◽  
Platelet Function ◽  
Association Studies ◽  
Interindividual Variation ◽  
Genome Wide Association Studies ◽  
Receptor Kinase ◽  
G Protein Coupled Receptor ◽  
Functional Responses ◽  
Glycoprotein Vi ◽  
G Protein Coupled

The interindividual variation in the functional response of platelets to activation by agonists is heritable. Genome-wide association studies (GWAS) of quantitative measures of platelet function have thus far identified fewer than 20 distinctly associated variants, some with unknown mechanisms. Here, we report GWAS of pathway specific functional responses to agonism by ADP, a glycoprotein VI-specific collagen mimetic and thrombin receptor-agonist peptides, each specific to one of the G protein-coupled receptors PAR-1 and PAR-4, in subsets of 1,562 individuals. We identified an association (P=2.75x10-40) between a common intronic variant, rs10886430 in the G protein-coupled receptor kinase 5 gene (GRK5), and the sensitivity of platelets to activate through PAR-1. The variant resides in a megakaryocyte-specific enhancer bound by the transcription factors GATA1 and MEIS1. The minor allele (G) is associated with fewer GRK5 transcripts in platelets and greater sensitivity of platelets to activate through PAR-1. We show that thrombin mediated activation of human platelets causes binding of GRK5 to PAR-1 and that deletion of the mouse homologue Grk5 enhances thrombin induced platelet activation sensitivity and increases platelet accumulation at the site of vascular injury. This corroborates evidence that the human G-allele of rs10886430 associates with greater risks of cardiovascular diseases. In summary, by combining the results of pathway specific GWAS and eQTL studies in humans with the results of platelet function studies in Grk5-/- mice, we obtain evidence that GRK5 regulates the human platelet response to thrombin via the PAR-1 pathway.

NAADP regulates human platelet function

2011 ◽  
Vol 441 (1) ◽  
pp. 435-442 ◽  
Author(s):  
Carmen H. Coxon ◽  
Alexander M. Lewis ◽  
Amanda J. Sadler ◽  
Sridhar R. Vasudevan ◽  
Andrew Thomas ◽  
...  
Keyword(s):  
Receptor Antagonist ◽  
Binding Site ◽  
Platelet Activation ◽  
Platelet Function ◽  
Cell Activation ◽  
Radioligand Binding ◽  
Human Platelet ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Glycoprotein Vi

Platelets play a vital role in maintaining haemostasis. Human platelet activation depends on Ca2+ release, leading to cell activation, granule secretion and aggregation. NAADP (nicotinic acid–adenine dinucleotide phosphate) is a Ca2+-releasing second messenger that acts on acidic Ca2+ stores and is used by a number of mammalian systems. In human platelets, NAADP has been shown to release Ca2+ in permeabilized human platelets and contribute to thrombin-mediated platelet activation. In the present study, we have further characterized NAADP-mediated Ca2+ release in human platelets in response to both thrombin and the GPVI (glycoprotein VI)-specific agonist CRP (collagen-related peptide). Using a radioligand-binding assay, we reveal an NAADP-binding site in human platelets, indicative of a platelet NAADP receptor. We also found that NAADP releases loaded 45Ca2+ from intracellular stores and that total platelet Ca2+ release is inhibited by the proton ionophore nigericin. Ned-19, a novel cell-permeant NAADP receptor antagonist, competes for the NAADP-binding site in platelets and can inhibit both thrombin- and CRP-induced Ca2+ release in human platelets. Ned-19 has an inhibitory effect on platelet aggregation, secretion and spreading. In addition, Ned-19 extends the clotting time in whole-blood samples. We conclude that NAADP plays an important role in human platelet function. Furthermore, the development of Ned-19 as an NAADP receptor antagonist provides a potential avenue for platelet-targeted therapy and the regulation of thrombosis.

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

1998 ◽  
Vol 80 (08) ◽  
pp. 310-315 ◽  
Author(s):  
Marie-Christine Bouton ◽  
Christophe Thurieau ◽  
Marie-Claude Guillin ◽  
Martine Jandrot-Perrus
Keyword(s):  
Platelet Activation ◽  
Electrostatic Interactions ◽  
Platelet Glycoprotein ◽  
Thrombin Receptor ◽  
Central Position ◽  
Amidolytic Activity ◽  
N Terminus ◽  
Hydrolysis Of ◽  
Chemical Cross Linking ◽  
Positively Charged

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.

Preparation and properties of three analogues of [5-leucine]enkephalin, substrates for enzyme conversion studies

1985 ◽  
Vol 50 (6) ◽  
pp. 1329-1334
Author(s):  
Jaroslav Vičar ◽  
Linda Servítová ◽  
Martin Flegel ◽  
Karel Hauzer ◽  
Tomislav Barth
Keyword(s):  
High Pressure ◽  
Liquid Chromatography ◽  
Guinea Pig ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Guinea Pig Ileum ◽  
Opioid Activity ◽  
C Terminus ◽  
N Terminus ◽  
Fragment Condensation

Analogues of [5-Leu]enkephalin, prolonged by methionine on the N-terminus or, by lysine or methionine on the C-terminus were prepared by fragment condensation, purified by ion exchange chromatography or high-pressure liquid chromatography. The substances were characterised by their opioid activity in a test on guinea-pig ileum in comparison with the activity of [5-Leu]enkephalin.

Impaired Platelet Function in Sept8-Deficient Mice In Vitro

2020 ◽  
Author(s):  
Kerstin Jurk ◽  
Katharina Neubauer ◽  
Victoria Petermann ◽  
Elena Kumm ◽  
Barbara Zieger
Keyword(s):  
Platelet Function ◽  
Thrombin Generation ◽  
Human Platelets ◽  
Platelet Surface ◽  
Integrin Αiibβ3 ◽  
Glycoprotein Vi ◽  
Fibrinogen Receptor ◽  
Static Conditions ◽  
The Impact

AbstractSeptins (Septs) are a widely expressed protein family of 13 mammalian members, recognized as a unique component of the cytoskeleton. In human platelets, we previously described that SEPT4 and SEPT8 are localized surrounding α-granules and move to the platelet surface after activation, indicating a possible role in platelet physiology. In this study, we investigated the impact of Sept8 on platelet function in vitro using Sept8-deficient mouse platelets. Deletion of Sept8 in mouse platelets caused a pronounced defect in activation of the fibrinogen receptor integrin αIIbβ3, α-granule exocytosis, and aggregation, especially in response to the glycoprotein VI agonist convulxin. In contrast, δ-granule and lysosome exocytosis of Sept8-deficient platelets was comparable to wild-type platelets. Sept8-deficient platelet binding to immobilized fibrinogen under static conditions was diminished and spreading delayed. The procoagulant activity of Sept8-deficient platelets was reduced in response to convulxin as determined by lactadherin binding. Also thrombin generation was decreased relative to controls. Thus, Sept8 is required for efficient integrin αIIbβ3 activation, α-granule release, platelet aggregation, and contributes to platelet-dependent thrombin generation. These results revealed Sept8 as a modulator of distinct platelet functions involved in primary and secondary hemostatic processes.

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