Antibodies to Protease-Activated Receptor 3 Inhibit Activation of Mouse Platelets by Thrombin

Blood ◽  
1998 ◽  
Vol 91 (11) ◽  
pp. 4152-4157 ◽  
Author(s):  
Hiroaki Ishihara ◽  
Dewan Zeng ◽  
Andrew J. Connolly ◽  
Carmen Tam ◽  
Shaun R. Coughlin
Keyword(s):  
Platelet Activation ◽  
Receptor Agonist ◽  
Thrombin Receptor ◽  
Definitive Evidence ◽  
Mouse Homologue ◽  
Thromboxane Receptor ◽  
Protease Activated Receptor 1

Recent studies of mice deficient in the thrombin receptor, protease-activated receptor 1 (PAR1), provided definitive evidence for the existence of a second thrombin receptor in mouse platelets. We recently identified a new thrombin receptor designated protease-activated receptor 3 (PAR3). The mRNA encoding a mouse homologue of PAR3 was highly expressed in mouse splenic megakaryocytes, making it a good candidate for the missing mouse platelet thrombin receptor. We now report that PAR3 protein is expressed on the surface of mouse platelets and that PAR3 antibodies partially inhibit activation of mouse platelets by thrombin but not U46619, a thromboxane receptor agonist. These observations suggest that PAR3 contributes to mouse platelet activation by thrombin.

Antibodies to Protease-Activated Receptor 3 Inhibit Activation of Mouse Platelets by Thrombin

Blood ◽  
1998 ◽  
Vol 91 (11) ◽  
pp. 4152-4157 ◽  
Author(s):  
Hiroaki Ishihara ◽  
Dewan Zeng ◽  
Andrew J. Connolly ◽  
Carmen Tam ◽  
Shaun R. Coughlin
Keyword(s):  
Platelet Activation ◽  
Receptor Agonist ◽  
Thrombin Receptor ◽  
Definitive Evidence ◽  
Mouse Homologue ◽  
Thromboxane Receptor ◽  
Protease Activated Receptor 1

Abstract Recent studies of mice deficient in the thrombin receptor, protease-activated receptor 1 (PAR1), provided definitive evidence for the existence of a second thrombin receptor in mouse platelets. We recently identified a new thrombin receptor designated protease-activated receptor 3 (PAR3). The mRNA encoding a mouse homologue of PAR3 was highly expressed in mouse splenic megakaryocytes, making it a good candidate for the missing mouse platelet thrombin receptor. We now report that PAR3 protein is expressed on the surface of mouse platelets and that PAR3 antibodies partially inhibit activation of mouse platelets by thrombin but not U46619, a thromboxane receptor agonist. These observations suggest that PAR3 contributes to mouse platelet activation by thrombin.

Extracellular Mutations of Protease-Activated Receptor-1 Result in Differential Activation by Thrombin and Thrombin Receptor Agonist Peptide

2000 ◽  
Vol 58 (6) ◽  
pp. 1178-1187 ◽  
Author(s):  
Brian D. Blackhart ◽  
Lily Ruslim-Litrus ◽  
Chin-Chun Lu ◽  
Veronica L. Alves ◽  
Willy Teng ◽  
...  
Keyword(s):  
Receptor Agonist ◽  
Thrombin Receptor ◽  
Differential Activation ◽  
Agonist Peptide ◽  
Protease Activated Receptor 1

Identification of a Novel 33-kDa Ser/Thr Kinase that Phosphorylates the Cytoplasmic Tail of Protease-activated Receptor 1 (Thrombin Receptor) in Human Platelets

2000 ◽  
Vol 83 (04) ◽  
pp. 617-621 ◽  
Author(s):  
Masaru Ido ◽  
Tatsuya Hayashi ◽  
Esteban Gabazza ◽  
Koji Suzuki
Keyword(s):  
Protein Kinase ◽  
Receptor Agonist ◽  
Thromboxane A2 ◽  
Cytoplasmic Tail ◽  
Fold Increase ◽  
Human Platelets ◽  
Thrombin Receptor ◽  
Glutathione S Transferase ◽  
Protease Activated Receptor 1 ◽  
Stimulation Of

SummaryStimulation of human platelets with thrombin or thrombin receptor agonist peptide (TRAP/ Ser-Phe-Leu-Leu-Arg-Asn) resulted in phosphorylation of the protease-activated receptor 1 (PAR1). However, protein kinase(s), capable of phosphorylating PAR1 upon activation of this receptor, has not been as yet identified in human platelets. The present study was undertaken to assess the presence of protein kinase(s) that may interact with PAR1 using a procedure based on the ability of protein kinase to undergo renaturation and phosphorylate a protein substrate fixed in a gel. We employed a fusion protein that was prepared using a glutathione S-transferase (GST) and the cytoplasmic tail of PAR1 (Pro368-Thr425)(GST-PAR1) or a reverse sequenced peptide of this domain (GST-rPAR1). The results showed that treatment of platelets with thrombin induced about 10-fold increase in the activity of the 33-kDa Ser/Thr protein kinase, which was also activated by TRAP, but not by hirudin-treated thrombin or diisopropylfluorophosphate-inactivated thrombin, suggesting that it is activated through PAR1. Furthermore, treatment of platelets with thromboxane A2 analog, STA2, led to an activation of this protein kinase and phosphorylation of PAR1. In conclusion, the present study provides evidence of homologous and heterologous activation of a novel 33-kDa Ser/Thr kinase that phosphorylates the cytoplasmic tail of PAR1.

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.

Aprotinin: is it prothrombotic?

Perfusion ◽  
2001 ◽  
Vol 16 (5) ◽  
pp. 401-409 ◽  
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.

Abstract 16292: Selective Platelet Protease-Activated-Receptor (PAR)1 and PAR4 Mediated Thrombin Desensitisation in the Elderly is Correlated With Inflammation and Chronic Thrombin Receptor Stimulation

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Sonali R Gnanenthiran ◽  
Gabrielle Pennings ◽  
Caroline Reddel ◽  
Heather Campbell ◽  
Justin Hamilton ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Platelet Activation ◽  
The Elderly ◽  
Elderly Subjects ◽  
Thrombin Receptor ◽  
Receptor Stimulation ◽  
Activation Markers ◽  
Tnf Α ◽  
D Dimer ◽  
Granule Release

Introduction: Platelet activation, by adenosine diphosphate (ADP) via P2Y 12 receptors and thrombin via PAR1 and PAR4, is a key therapeutic target in cardiovascular disease (CVD). The efficacy of antiplatelet agents diminishes in the elderly, but it is unknown whether these pathways change with aging. Hypothesis: Platelet activation pathways change with aging. Methods: Platelet activity was evaluated in young (20-30yrs), middle-aged (40-55yrs) and elderly (≥70yrs) healthy volunteers (n=174). Whole blood aggregometry and flow cytometry (P-selectin: α-granule release; CD63: dense granule release; PAC1 binding: activated GPIIb/IIIa) were performed under basal conditions and post ex vivo stimulation with ADP, thrombin, PAR1 agonist or PAR4 agonist. EC 50 and E max values were derived for each agonist. Receptor cleavage and quantification (P2Y 12 ; PAR1; PAR4; GPIbα) were assessed with flow cytometry. Thrombin generation (D-Dimer) and inflammation (interleukin [IL]-1β; tumour necrosis factor [TNF]-α) were assessed via ELISA. Results: The elderly had higher basal platelet activation markers (P-selectin, CD63, activated GPIIb/IIIa) than the young, with higher basal activity correlating with increasing IL-1β. P2Y 12 receptor density was higher in the elderly and associated with greater ADP-induced platelet aggregation and activation. Elderly subjects had less platelet activation in response to thrombin (higher EC 50 ), demonstrating hyporeactivity to selective stimulation of PAR1 or PAR4, more basal PAR1/PAR4 cleavage, and less inducible PAR1/PAR4 cleavage. This was associated with reduced thrombin binding receptor GPIbα and reduced secondary ADP contribution to thrombin-mediated activation. D-Dimer and TNF-α levels were elevated in the elderly, and inversely correlated with platelet thrombin sensitivity, implying a role of desensitization from chronic thrombin receptor stimulation. Conclusion: Aging is associated with increased basal platelet activation and hyperreactivity to ADP, but selective desensitization to thrombin. The latter appears mediated by chronic thrombin receptor stimulation and inflammation. Age-specific antiplatelet strategies may require selective targeting of these pathways to treat CVD in the elderly.

Structure-function relationships in the thrombin receptor agonist peptides

Peptides ◽  
1993 ◽  
pp. 141-142 ◽  
Author(s):  
Dong-Mei Feng ◽  
Daniel F. Veber ◽  
Tom Connolly ◽  
Ruth F. Nutt
Keyword(s):  
Structure Function ◽  
Receptor Agonist ◽  
Thrombin Receptor
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