Platelets from children are hyper-responsive to activation by thrombin receptor activator peptide and adenosine diphosphate compared to platelets from adults

AbstractCardiopulmonary bypass (CPB) is associated with platelet dysfunction (PD), an important cause of postoperative bleeding. The etiology of PD is not completely understood. We mapped the platelets' function during CPB to determine the etiology of PD. Platelets activation, measured by procaspase activating compound-1 and P-selectin expression (CD62P), after activation by adenosine diphosphate and thrombin receptor activator peptide, were decreased by protamine. Changes during CPB were insignificant. Platelet-leukocyte aggregation was increased by CPB but not by protamine. Platelet apoptosis marker, annexin V, was increased by protamine. Changes during CPB were insignificant. Our findings demonstrate that protamine given after CPB plays a central role in PD and count decrease.

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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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Rabbit and rat platelets do not respond to thrombin receptor peptides that activate human platelets

Blood ◽

10.1182/blood.v82.1.103.bloodjournal821103 ◽

1993 ◽

Vol 82 (1) ◽

pp. 103-106 ◽

Cited By ~ 53

Author(s):

RL Kinlough-Rathbone ◽

ML Rand ◽

MA Packham

Keyword(s):

Guinea Pig ◽

Shape Change ◽

Adenosine Diphosphate ◽

Human Platelets ◽

Dense Granule ◽

Thrombin Receptor ◽

Amino Acid Residues ◽

Reversible Aggregation ◽

Human Thrombin ◽

Rat Platelets

Human platelets are aggregated and induced to release their granule contents and form thromboxane by peptides as short as 6-amino acid residues (SFLLRN) corresponding to the newly released N-terminus of the thrombin receptor that is cleaved by thrombin. Using washed platelets, we found that these responses to SFLLRN (2 to 6 mumol/L) were enhanced by fibrinogen. However, neither SFLLRN nor SFLLRNPNDKYEPF had any effect on washed rabbit or rat platelets, although they were fully responsive to human thrombin. Concentrations of the peptides as high as 100 mumol/L did not cause the platelets of rabbits or rats to change shape, aggregate, release granule contents, or form thromboxane. SFLLRN did not affect the extent of aggregation induced by adenosine diphosphate (ADP) or a low concentration of thrombin. Pig platelets responded to 50 mumol/L SFLLRN with reversible aggregation, which was enhanced by fibrinogen, but not accompanied by the release of dense granule contents. Guinea pig platelets aggregated and released granule contents in response to 25 or 50 mumol/L of SFLLRN, but responded with only shape change to lower concentrations. Thus, these experiments indicate that rabbit and rat platelets lack a functional response to human thrombin receptor peptides that fully activate the previously described human thrombin receptor, despite a full response of both rabbit and rat platelets to human thrombin, and that pig and guinea pig platelets have incomplete responses to these human thrombin receptor peptides. The results suggest that platelets of rabbits and rats, and perhaps guinea pigs and pigs, respond to thrombin through an alternative receptor that has also been suggested to be present on human platelets.

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Singlet Oxygen Inhibits Agonist-Induced P-Selectin Expression and Formation of Platelet Aggregates

Clinical and Applied Thrombosis/Hemostasis ◽

10.1177/107602960100700307 ◽

2001 ◽

Vol 7 (3) ◽

pp. 219-224 ◽

Cited By ~ 3

Author(s):

T.W. Stief ◽

W.P. Jeske ◽

J. Walenga ◽

C. Schultz ◽

V. Kretschmer ◽

...

Keyword(s):

Singlet Oxygen ◽

Control Level ◽

Adenosine Diphosphate ◽

Saline Control ◽

Radical Scavenger ◽

Oxidant Concentration ◽

Receptor Activator ◽

Chloramine T ◽

Platelet Aggregates ◽

Signal Action

Major mediators of activated polymorphonuclear leukocytes (PMN) are the oxidants HOCl and chloramine, which are a source for the nonradical photon-emitting oxidant singlet oxygen (1O2). We were interested in a possible platelet-modulating activity of 1O2, As a stable 1O2 source we chose the mild oxidant chloramine T® (CT), which mimics the natural chloramine N-chloro-taurine. Freshly drawn native whole blood from donors (n = 5) was incubated at 0 to 3 mM CT for I minute at 37°C. Then saline, 10 μM adenosine diphosphate (ADP), 5 μg/mL collagen, or 6.25 μM thrombin receptor activator peptide (TRAP) were added and the mixtures were allowed to incubate for 3 minutes at 37°C. Aliquots of activated blood were fixed in 1% para-formaldehyde. After removal of the fixative, platelets were labeled with anti-CD61-FITC and anti-CD62P-PE antibodies and analyzed by flow cytometry. An oxidant concentration-dependent decrease in the expression of -selectin appeared (at 3 mM CT to 39, 23, and 20% of the 100% saline control level for ADP, collagen, and TRAP, respectively). There was also an oxidant concentration-dependent decrease in the formation of platelet aggregates (at 3 mM CT to 8, 12, and 13% of the 100% saline control level for ADP, collagen, and TRAP, respectively; the 50% effective dose was 1.0 to 1.5 mM chloramine). In ADP- and TRAP-stimulated platelets, an oxidant-mediated increase in platelet fragments appeared (at 3 mM CT: three- to fourfold of the initial value). The addition to the blood of 30 mM of the oxy-radical scavenger mannitol in contrast to excess methionine did not antagonize these oxidative modulations of platelet activation. The results were confirmed using equimolar concentrations of NaOCl and N-chloro-taurine. This study shows that 1O2 inhibits platelets, decreasing the expression of CD62P and the formation of platelet aggregates. Activated PMN might modulate hemostasis, shifting it into an antithrombotic state. The physiologic signal action and the direct anticoagulant action of 1O2 (released by chloramines such as vancomycin) might be a new principle for pharmacologic intervention in atherothrombosis.

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Whole blood platelet aggregation kinetics under cardiopulmonary bypass: A pilot study

The International Journal of Artificial Organs ◽

10.1177/0391398819882440 ◽

2019 ◽

Vol 43 (3) ◽

pp. 208-214

Author(s):

Tobias Petzold ◽

Erik Bagaev ◽

Helen Herzog ◽

Frank Born ◽

Dominik Hoechter ◽

...

Keyword(s):

Cardiopulmonary Bypass ◽

Platelet Aggregation ◽

Life Support ◽

Platelet Reactivity ◽

Extracorporeal Life Support ◽

Adenosine Diphosphate ◽

Thrombin Receptor ◽

Electrode Impedance ◽

Platelet Counts ◽

Impedance Aggregometry

Assessing the platelets’ functional status during surgery on cardiopulmonary bypass is challenging. This study used multiple electrode impedance aggregometry (Multiplate®) to create a timeline of platelet aggregation changes as induced by cardiopulmonary bypass in antiplatelet-naive patients undergoing elective surgery for mitral valve regurgitation. We performed six consecutive measurements (T1: pre-operatively, T2: after heparinization, T3: 3 min after establishment of cardiopulmonary bypass, T4: immediately after administration of cardioplegia, T5: 5 min after administration of cardioplegia, and T6: 45 min after administration of cardioplegia). Platelet aggregation was determined after stimulation with 3.2-μg/mL collagen, 6.4-μM adenosine diphosphate, and 32-μM thrombin receptor activating peptide. Five patients were included (age: 64 ± 10 years, one female). We observed a decrease in hematocrit levels by −17.1% ± 3.7% (T1 vs T6) with a drop after establishment of cardiopulmonary bypass (T2 vs T3) and slightly decreasing platelet counts by −6.2% ± 7.7% (T1 vs T6). Immediately after establishment of cardiopulmonary bypass (T2 vs T3), we observed reduced platelet aggregation responses for stimulation with adenosine diphosphate (−19.7% ± 12.8%) and thrombin receptor activating peptide (−19.3% ± 6.3%). Interestingly, we found augmented platelet aggregation for all stimuli 45 min after administration of cardioplegia (T5 vs T6) with the strongest increase for collagen (+83.4% ± 42.8%; adenosine diphosphate: +39.0% ± 37.2%; thrombin receptor activating peptide: +34.5% ± 18.5%). Thus, after an initial drop due to hemodilution upon establishment of cardiopulmonary bypass, platelet reactivity increased over time which was not outweighed by decreasing platelet counts due to mechanical platelet destruction and absorption. These findings have implications for rational transfusion, peri-operative antiplatelet therapy, and for the management of patients on other extracorporeal support, such as extracorporeal life support or extracorporeal membrane oxygenation.

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Increased platelet aggregation and in vivo platelet activation after granulocyte colony-stimulating factor administration

Thrombosis and Haemostasis ◽

10.1160/th10-08-0530 ◽

2011 ◽

Vol 105 (04) ◽

pp. 655-662 ◽

Cited By ~ 32

Author(s):

Alexander Spiel ◽

Johann Bartko ◽

Michael Schwameis ◽

Christa Firbas ◽

Jolanta Siller-Matula ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Controlled Trial ◽

Adenosine Diphosphate ◽

Thrombin Receptor ◽

Granulocyte Colony Stimulating Factor ◽

Colony Stimulating Factor ◽

Neutrophil Recovery ◽

Stimulating Factor

SummaryGranulocyte colony-stimulating factor (G-CSF) stimulates the bone marrow to produce granulocytes and stem cells and is widely used to accelerate neutrophil recovery after chemotherapy. Interestingly, specific G-CSF receptors have been demonstrated not only on myeloid cells, but also on platelets. Data on the effects of G-CSF on platelet function are limited and partly conflicting. The objective of this study was to determine the effect of G-CSF on platelet aggregation and in vivo platelet activation. Seventy-eight, healthy volunteers were enrolled into this randomised, placebo-controlled trial. Subjects received 5 μg/kg methionyl human granulocyte colony-stimulating factor (r-metHuG-CSF, filgrastim) or placebo subcutaneously for four days. We determined platelet aggregation with a whole blood impedance aggregometer with various, clinically relevant platelet agonists (adenosine diphosphate [ADP], collagen, arachidonic acid [AA], ristocetin and thrombin receptor activating peptide 6 [TRAP]). Filgrastim injection significantly enhanced ADP (+40%), collagen (+60%) and AA (+75%) -induced platelet aggregation (all p<0.01 as compared to placebo and p<0.001 as compared to baseline). In addition, G-CSF enhanced ristocetin-induced platelet aggregation (+18%) whereas TRAP-induced platelet aggregation decreased slightly (-14%) in response to filgrastim. While baseline aggregation with all agonists was only slightly but insignificantly higher in women than in men, this sex difference was enhanced by G-CSF treatment, and became most pronounced for ADP after five days (p<0.001). Enhanced platelet aggregation translated into a 75% increase in platelet activation as measured by circulating soluble P-selectin. G-CSF enhances platelet aggregation and activation in humans. This may put patients suffering from cardiovascular disease and cancer at risk for thrombotic events.

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Differential inhibition of platelet aggregation induced by adenosine diphosphate or a thrombin receptor-activating peptide in patients treated with bolus chimeric 7E3 Fab: Implications for inhibition of the internal pool of GPIIb/IIIa receptors

Journal of the American College of Cardiology ◽

10.1016/0735-1097(95)00391-6 ◽

1995 ◽

Vol 26 (7) ◽

pp. 1665-1671 ◽

Cited By ~ 111

Author(s):

Neal S. Kleiman ◽

Albert E. Raizner ◽

Robert Jordan ◽

Ann L. Wang ◽

Daniel Norton ◽

...

Keyword(s):

Platelet Aggregation ◽

Adenosine Diphosphate ◽

Thrombin Receptor ◽

Differential Inhibition ◽

Inhibition Of Platelet Aggregation

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Differential inhibition of adenosine diphosphate– versus thrombin receptor–activating peptide–stimulated platelet fibrinogen binding by abciximab due to different glycoprotein IIb/IIIa activation kinetics

Blood ◽

10.1182/blood.v98.5.1619 ◽

2001 ◽

Vol 98 (5) ◽

pp. 1619-1621 ◽

Cited By ~ 14

Author(s):

Artur-Aron Weber ◽

Karsten Schrör

Keyword(s):

Platelet Activation ◽

Adenosine Diphosphate ◽

Binding Kinetics ◽

Thrombin Receptor ◽

Differential Inhibition ◽

Activation Kinetics ◽

Binding Rate ◽

Fibrinogen Binding ◽

Activation Rate

The exposure of internal glycoprotein (GP) IIb/IIIa receptors has been proposed to explain the incomplete inhibition of aggregation of thrombin receptor–activating peptide (TRAP)-stimulated platelets by abciximab. However, a marked and rapid externalization of GPIIb/IIIa was also observed upon stimulation with 30 μM adenosine diphosphate (ADP). ADP-induced fibrinogen binding was completely inhibited by 10 μg/mL abciximab, 30 nM tirofiban, or 3 μg/mL eptifibatide, while fibrinogen binding induced by 100 μM TRAP was inhibited only by 50%. Interestingly, striking differences in fibrinogen binding kinetics in ADP- versus TRAP-stimulated platelets were observed. ADP-induced fibrinogen binding was much slower than that of abciximab. These differences in the fibrinogen binding rate were due to differential GPIIb/IIIa activation kinetics because the actual fibrinogen binding rate (measured by adding fibrinogen after platelet activation) was similar in ADP- and TRAP-stimulated platelets. Thus, the TRAP-induced GPIIb/IIIa activation rate would allow significant amounts of fibrinogen to occupy externalized GPIIb/IIIa receptors even in the presence of the inhibitor.

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Unfractionated Heparin Reduces the Anti-Platelet Effects of Abciximab but not Eptifibatide During PCI

Clinical and Applied Thrombosis/Hemostasis ◽

10.1177/1076029606293432 ◽

2006 ◽

Vol 12 (4) ◽

pp. 458-464 ◽

Cited By ~ 2

Author(s):

Efthymios N. Deliargyris ◽

Bharathi Upadhya ◽

Laura G. Melton ◽

Cheryl Thompson ◽

Melrose Fisher ◽

...

Keyword(s):

Unfractionated Heparin ◽

Receptor Agonist ◽

Adenosine Diphosphate ◽

Coronary Intervention ◽

Platelet Inhibition ◽

Thrombin Receptor ◽

Blood Samples ◽

Agonist Peptide ◽

Percutaneous Coronary ◽

Standard Weight

In 29 patients undergoing percutaneous coronary intervention (PCI), we obtained blood samples at baseline, 10 minutes after standard weight-based abciximab (n=15) or double-bolus eptifibatide (n=14) and 5 minutes after unfractionated heparin (UFH; 70 U/kg bolus). The median percent inhibition was significantly higher in the eptifibatide group compared with the abciximab group both before (96.5% [94-100] vs. 85% [77-89.5] [adenosine diphosphate; ADP]; 89.5% [84-95] vs. 59% [37.5-76.5] [thrombin receptor agonist peptide; TRAP], p<0.001 for both) and after UFH (95% [93-100] vs. 79% [68.8-87.5] [ADP]; 82% [77-93] vs. 51% [34.5-71.3] [TRAP], p<0.001 for both). Addition of UFH significantly reduced platelet inhibition in the abciximab group (85% [77-89.5] vs. 79% [68.8-87.5] [ADP]; 59% [37.5-76.5] vs. 51% [34.5-71.3] [TRAP], p<0.05 for both) but not in the eptifibatide group (96.5% [94-100] vs. 95% [93-100] [ADP]; 89.5% [84-95] vs. 82% [77-93] [TRAP], p=ns for both). Eptifibatide achieved superior platelet inhibition before but especially after UFH compared with abciximab.

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The small proteoglycan decorin supports adhesion and activation of human platelets

Blood ◽

10.1182/blood.v100.5.1707.h81702001707_1707_1714 ◽

2002 ◽

Vol 100 (5) ◽

pp. 1707-1714 ◽

Cited By ~ 36

Author(s):

Gianni Guidetti ◽

Alessandra Bertoni ◽

Manuela Viola ◽

Enrica Tira ◽

Cesare Balduini ◽

...

Keyword(s):

Tyrosine Phosphorylation ◽

Platelet Adhesion ◽

Adenosine Diphosphate ◽

Human Platelets ◽

Membrane Receptors ◽

Thrombin Receptor ◽

Membrane Glycoprotein ◽

Intracellular Proteins ◽

Subendothelial Matrix ◽

Small Proteoglycan

Decorin is a small leucine-rich proteoglycan able to interact with several molecules of the subendothelial matrix, such as collagen and fibronectin. In this work, we investigated the ability of purified decorin to support adhesion of human platelets. We found that gel-filtered platelets were actually able to interact with immobilized decorin. Platelet adhesion to decorin was time dependent, required the presence of Mg2+ ions, and was totally mediated by the protein core of the proteoglycan. Platelet stimulation with either adenosine diphosphate (ADP) or a thrombin receptor–activating peptide significantly increased interaction of these cells with the proteoglycan. Upon adhesion to immobilized decorin a number of platelet proteins were found to become tyrosine-phosphorylated. By immunoprecipitation experiments with specific antibodies, the tyrosine phosphorylation of the tyrosine kinase Syk and the phospholipase Cγ2 (PLCγ2) isozyme was demonstrated in decorin-adherent platelets. Interaction of platelets with decorin was selectively prevented by 2 different antibodies against membrane integrin α2β1, but not by a number of antibodies against other membrane receptors. In addition, integrin α2β1, purified from platelet membranes, was able to specifically interact with immobilized decorin. Finally, purified decorin bound to Sepharose beads could precipitate integrin α2β1 from a platelet membrane glycoprotein preparation. Therefore, these results demonstrate that human platelets can bind to immobilized decorin through integrin α2β1, and that this interaction results in the tyrosine phosphorylation of intracellular proteins.

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