scholarly journals The Impact of Cold Storage on Adenosine Diphosphate-Mediated Platelet Responsiveness

TH Open ◽  
2020 ◽  
Vol 04 (03) ◽  
pp. e163-e172
Author(s):  
Juergen Koessler ◽  
Philipp Klingler ◽  
Marius Niklaus ◽  
Katja Weber ◽  
Angela Koessler ◽  
...  
Keyword(s):  
Cold Storage ◽  
Whole Blood ◽  
Room Temperature ◽  
Purinergic Receptor ◽  
Adenosine Diphosphate ◽  
Receptor Expression ◽  
Activation Markers ◽  
Inhibitory Pathways ◽  
The Impact ◽  
Induced Aggregation

Abstract Introduction Cold storage of platelets is considered to contribute to lower risk of bacterial growth and to more efficient hemostatic capacity. For the optimization of storage strategies, it is required to further elucidate the influence of refrigeration on platelet integrity. This study focused on adenosine diphosphate (ADP)-related platelet responsiveness. Materials and Methods Platelets were prepared from apheresis-derived platelet concentrates or from peripheral whole blood, stored either at room temperature or at 4°C. ADP-induced aggregation was tested with light transmission. Activation markers, purinergic receptor expression, and P2Y12 receptor function were determined by flow cytometry. P2Y1 and P2X1 function was assessed by fluorescence assays, cyclic nucleotide concentrations by immunoassays, and vasodilator-stimulated phosphoprotein (VASP)-phosphorylation levels by Western blot analysis. Results In contrast to room temperature, ADP-induced aggregation was maintained under cold storage for 6 days, associated with elevated activation markers like fibrinogen binding or CD62P expression. Purinergic receptor expression was not essentially different, whereas P2Y1 function deteriorated rapidly at cold storage, but not P2Y12 activity. Inhibitory pathways of cold-stored platelets were characterized by reduced responses to nitric oxide and prostaglandin E1. Refrigeration of citrated whole blood also led to the attenuation of induced inhibition of platelet aggregation, detectable within 24 hours. Conclusion ADP responsiveness is preserved under cold storage for 6 days due to stable P2Y12 activity and concomitant disintegration of inhibitory pathways enabling a higher reactivity of stored platelets. The ideal storage time at cold temperature for the highest hemostatic effect of platelets should be evaluated in further studies.

Aspirin-Dependent Effects on Purinergic P2Y1 Receptor Expression

2019 ◽  
Vol 119 (05) ◽  
pp. 726-734 ◽  
Author(s):  
Isabella Massimi ◽  
Laura Alemanno ◽  
Maria Guarino ◽  
Raffaella Guerriero ◽  
Massimo Mancone ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Thromboxane A2 ◽  
Adenosine Diphosphate ◽  
Receptor Expression ◽  
P2y1 Receptor ◽  
Biochemical Pathway ◽  
Thromboxane A2 Receptor ◽  
Induced Aggregation

AbstractChronic treatment with aspirin in healthy volunteers (HVs) is associated with recovery of adenosine diphosphate (ADP)-induced platelet activation. The purinergic P2Y1 receptor exerts its effects via a Gq-protein, which is the same biochemical pathway activated by thromboxane-A2 receptor. We hypothesized that recovery of ADP-induced platelet activation could be attributed to increased P2Y1 expression induced by chronic aspirin exposure. We performed a multi-phase investigation which embraced both in vitro and in vivo experiments conducted in (1) human megakaryoblastic DAMI cells, (2) human megakaryocytic progenitor cell cultures, (3) platelets obtained from HVs treated with aspirin and (4) platelets obtained from aspirin-treated patients. DAMI cells treated with aspirin or WY14643 (PPARα agonist) had a significant up-regulation of P2Y1 mRNA, which was shown to be a PPARα-dependent process. In human megakaryocytic progenitors, in the presence of aspirin or WY14643, P2Y1 mRNA expression was higher than in mock culture. P2Y1 expression increased in platelets obtained from HVs treated with aspirin for 8 weeks. Platelets obtained from patients who were on aspirin for more than 2 months had increased P2Y1 expression and ADP-induced aggregation compared with patients on aspirin treatment for less than a month. Overall, our results suggest that aspirin induces genomic changes in megakaryocytes leading to P2Y1 up-regulation and that PPARα is the nuclear receptor involved in this regulation. Since P2Y1 is coupled to the same Gq-protein of thromboxane-A2 receptor, platelet adaptation in response to pharmacological inhibition seems not to be receptor specific, but may involve other receptors with the same biochemical pathway.

scholarly journals Hypoxia Modulates Platelet Purinergic Signalling Pathways

2019 ◽  
Vol 120 (02) ◽  
pp. 253-261 ◽  
Author(s):  
Gordon G. Paterson ◽  
Jason M. Young ◽  
Joseph A. Willson ◽  
Christopher J. Graham ◽  
Rebecca C. Dru ◽  
...  
Keyword(s):  
High Altitude ◽  
Sea Level ◽  
Platelet Reactivity ◽  
Clinical Importance ◽  
Adenosine Diphosphate ◽  
Purinergic Signalling ◽  
Systematic Assessment ◽  
Vasp Phosphorylation ◽  
The Impact ◽  
Induced Aggregation

Abstract Background Hypoxia resulting from ascent to high-altitude or pathological states at sea level is known to increase platelet reactivity. Previous work from our group has suggested that this may be adenosine diphosphate (ADP)-specific. Given the clinical importance of drugs targeting ADP pathways, research into the impact of hypoxia on platelet ADP pathways is highly important. Methods Optimul aggregometry was performed on plasma from 29 lowland residents ascending to 4,700 m, allowing systematic assessment of platelet reactivity in response to several platelet agonists. Aggregometry was also performed in response to ADP in the presence of inhibitors of the two main ADP receptors, P2Y1 and P2Y12 (MRS2500 and cangrelor, respectively). Phosphorylation of vasodilator-stimulated phosphoprotein (VASP), a key determinant of platelet aggregation, was analysed using the VASPFix assay. Results Hypobaric hypoxia significantly reduced the ability of a fixed concentration of cangrelor to inhibit ADP-induced aggregation and increased basal VASP phosphorylation. However, in the absence of P2Y receptor inhibitors, we did not find evidence of increased platelet sensitivity to any of the agonists tested and found reduced sensitivity to thrombin receptor-activating peptide-6 amide. Conclusion Our results provide evidence of increased P2Y1 receptor activity at high altitude and suggest down-regulation of the P2Y12 pathway through increased VASP phosphorylation. These changes in ADP pathway activity are of potential therapeutic significance to high-altitude sojourners and hypoxic sea level patients prescribed platelet inhibitors and warrant further investigation.

scholarly journals Role of Purinergic Receptor Expression and Function for Reduced Responsiveness to Adenosine Diphosphate in Washed Human Platelets

PLoS ONE ◽  
2016 ◽  
Vol 11 (1) ◽  
pp. e0147370 ◽  
Author(s):  
Juergen Koessler ◽  
Stephanie Hermann ◽  
Katja Weber ◽  
Angela Koessler ◽  
Sabine Kuhn ◽  
...  
Keyword(s):  
Purinergic Receptor ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Receptor Expression ◽  
And Function

Leukocytes Can Enhance Platelet-mediated Aggregation and Thromboxane Release via  Interaction of P-selectin Glycoprotein Ligand 1 with P-selectin

2001 ◽  
Vol 94 (1) ◽  
pp. 145-151 ◽  
Author(s):  
Nauder Faraday ◽  
Robert B. Scharpf ◽  
Jeffrey M. Dodd-o ◽  
Elizabeth A. Martinez ◽  
Brian A. Rosenfeld ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Adenosine Triphosphate ◽  
Whole Blood ◽  
Platelet Rich Plasma ◽  
Adenosine Diphosphate ◽  
Live Cells ◽  
Vitro Analysis ◽  
Blocking Antibodies ◽  
Induced Aggregation

Background Platelet--leukocyte conjugates have been observed in patients with unstable coronary syndromes and after cardiopulmonary bypass. In vitro, the binding of platelet P-selectin to leukocyte P-selectin glycoprotein ligand-1 (PSGL1) mediates conjugate formation; however, the hemostatic implications of these cell--cell interactions are unknown. The aims of this study were to determine the ability of leukocytes to modulate platelet agonist--induced aggregation and secretion in the blood milieu, and to investigate the role of P-selectin and PSGL-1 in mediating these responses. Methods Blood was drawn from healthy volunteers for in vitro analysis of platelet agonist--induced aggregation, secretion (adenosine triphosphate, beta-thromboglobulin, and thromboxane), and platelet-leukocyte conjugate formation. Experiments were performed on live cells in whole blood or plasma to simulate physiologic conditions. Whole-blood impedance and optical aggregometry, flow cytometry, and enzyme-linked immunosorbent assays were performed in the presence and absence of blocking antibodies to P-selectin and PSGL1. The platelet-specific agonists, thrombin receptor activating peptide and adenosine diphosphate, were used to elicit platelet activation responses. Results Inhibition of platelet--leukocyte adherence by P- selectin and PSGL1 antibodies decreased agonist--induced aggregation in whole blood. The presence of leukocytes in platelet-rich plasma increased aggregation, and this increase was attenuated by P-selectin blocking antibodies. Data from flow cytometry confirmed that platelet-leukocyte conjugate formation contributed to aggregation responses. Blocking antibodies reduced platelet agonist--induced thromboxane release but had no impact on adenosine triphosphate and beta-thomboglobulin secretion. Conclusions Leukocytes can enhance platelet agonist--induced aggregation and thromboxane release in whole blood and platelet-rich plasma under shear conditions in vitro. Interaction of platelet P-selectin with leukocyte PSGL1 contributes substantially to these effects.

scholarly journals Antiaggregatory activity of new 1-ethylxanthine cyclohexylammonium salt in vitro

2013 ◽  
Vol 94 (5) ◽  
pp. 692-695
Author(s):  
F Kh Kamilov ◽  
G A Timirkhanova ◽  
A I Samorodova ◽  
A V Samorodov ◽  
F A Khaliullin ◽  
...  
Keyword(s):  
Acetic Acid ◽  
Platelet Aggregation ◽  
Aggregate Size ◽  
Adenosine Diphosphate ◽  
Biochemical Effect ◽  
Platelet Aggregate ◽  
Antiaggregatory Activity ◽  
The Impact ◽  
Induced Aggregation

Aim. To study the biochemical effect on hemostasis of a new cyclohexilammonium salt of 2-[1-ethyl-3-methyl-7(dioxotiethanyl-3)xantinyl-8-thio]acetic acid in vitro. Methods. Thromboelastography was performed using the citrate blood samples of healthy male donors. Global hemostatic effect, fibrinogen and platelet function, fibrinolysis and clot strength, and stability were analyzed at thromboelastography. The impact of firstly synthesized xantine derivative and pentoxifylline on the functional activity of platelets in vitro was studied using a laser analyzer of platelet aggregation. Adenosine diphosphate, collagen, epinephrine and ristocetin induced clotting were registered. General clotting characteristics, maximal aggregation values, maximal aggregation speed, mean platelet aggregate size, activity of platelet-derived factor 3, level of platelet-derived factor 4 were measured. Release of platelet-derived factors 3 and 4 at platelet aggregation were assessed after adenosinediphosphate-induced aggregation and centrifugation. Results. Cyclohexylammonium salt of 2-[1-ethyl-3-methyl-7-(dioxotiethanyl-3)xantinyl-8-thio]acetic acid in vitro showed antiaggregatory activity that exceeds such of pentoxifylline. It has been revealed that the second platelet aggregation wave, that is induced by small dose of adenosinediphosphate, is absent in the presence of the new cyclohexylammonium salt, lag-period in collagen-induced platelet aggregation elongates, and availability and release of platelet-derived factors 3 and 4 decreases. Conclusion. The research findings show potentially high antiaggregatory activity of 2-[1-ethyl-3-methyl-7-(dioxotiethanyl-3)xantinyl-8-thio]acetic acid cyclohexylammonium salt as an inhibitor of platelet release reaction.

scholarly journals The Effect of Tirofiban on Fibrinogen/Agonist-Induced Platelet Shape Change and Aggregation

2008 ◽  
Vol 14 (3) ◽  
pp. 295-302 ◽  
Author(s):  
I. Anita Jagroop ◽  
Dimitri P. Mikhailidis
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Whole Blood ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
Adenosine Diphosphate ◽  
Vascular Events ◽  
Spontaneous Platelet Aggregation ◽  
Induced Aggregation ◽  
Platelet Shape

There is evidence linking raised plasma fibrinogen (fib) and platelet hyperactivity with vascular events. One way to inhibit platelets is to block the platelet membrane glycoprotein (GP) IIb/IIIa receptor, which binds circulating fib or von Willebrand factor and cross-links platelets at the final common pathway to platelet aggregation. Tirofiban is a potent and specific fib receptor antagonist, used in the treatment of unstable angina. The authors assessed the effect of tirofiban on spontaneous platelet aggregation (SPA), fib-induced, serotonin (5HT)-induced, and adenosine diphosphate (ADP)-induced aggregation in whole blood by calculating the percentage free platelet count. These various agonists were used alone and in combination. The authors also measured the effect of tirofiban on agonists-induced (ADP, 5HT) platelet shape change (PSC). The effect of fib on PSC was also evaluated in platelet-rich plasma using a high-resolution (0.07 fL) channelyzer. Tirofiban significantly inhibited SPA, fib (2, 4, 8 g/L), ADP, ADP + fib combination, and 5HT-induced aggregation. Tirofiban had no effect on agonist-induced PSC. There was no apparent change in platelet volume with fib. In conclusion, tirofiban does not appear to have an effect on PSC, an early phase of platelet activation. Tirofiban seems to be a nonspecific and an effective inhibitor of platelet aggregation (a later phase of platelet activation) in whole blood. The clinical significance of these findings remains to be established.

Twice daily dosing of aspirin improves platelet inhibition in whole blood in patients with type 2 diabetes mellitus and micro- or macrovascular complications

2011 ◽  
Vol 106 (09) ◽  
pp. 491-499 ◽  
Author(s):  
Galia Spectre ◽  
Lisa Arnetz ◽  
Claes-Göran Östenson ◽  
Kerstin Brismar ◽  
Nailin Li ◽  
...  
Keyword(s):  
Diabetes Mellitus ◽  
Type 2 Diabetes ◽  
Type 2 Diabetes Mellitus ◽  
Whole Blood ◽  
Macrovascular Complications ◽  
Light Transmittance ◽  
Impedance Aggregometry ◽  
The Impact ◽  
Induced Aggregation

SummaryThe efficacy of low-dose aspirin in type 2 diabetes mellitus (T2DM) has been questioned. We tested if twice daily dosing of aspirin would be more effective in T2DM, possibly due to increased platelet turnover. A randomised cross-over study compared 75 mg aspirin OD, 75 mg BID and 320 mg OD (≥2 week treatment periods) in 25 patients with T2DM and micro- or macrovascular complications. Platelet responses were examined by impedance aggregometry (WBA) and the IMPACT-R aspirin test in whole blood, light transmittance aggregometry in plateletrich plasma (LTA), and urinary 11-dehydro-thromboxane B2 (TxM). Aspirin 75 mg BID decreased arachidonic acid (AA)-induced WBA compared to 75 mg OD (9.7 ± 4.5 vs. 12.6 ± 3.5 ohm; p=0.003) or to 320 mg OD (11.5 ± 4.2 Ohms; p=0.049). WBA responses to collagen were similarly attenuated by BID or high dosing (by 12–14%; p=0.02 for both). The IMPACT-R showed a better response to 75 mg BID compared to 75 mg OD (p=0.049), but not to 320 mg OD. AA-induced aggregation by LTA was <6.5% on all occasions, with no differences between aspirin dosages. TxM was reduced after 320 mg OD (p=0.002), but not 75 mg BID (p=0.07). Reticulated platelets were highly correlated with mean platelet volume (MPV; r2=0.74, p<0.0001). Both markers for platelet turnover were correlated with AA-induced WBA, but neither identified patients who benefited from BID dosing dependably. In conclusion, twice daily dosing improved laboratory responses to aspirin in high risk T2DM patients. Studies of whether BID dosing of aspirin can improve clinical outcomes in such patients are of interest.

scholarly journals Platelet Function in Paroxysmal Nocturnal Haemoglobinuria

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4884-4884
Author(s):  
Fathima Mohiyaddin Ayyalil ◽  
Samantha J Montague ◽  
Sarah Hicks ◽  
Amandeep Kaur ◽  
Anila Jahangiri ◽  
...  
Keyword(s):  
Platelet Count ◽  
Platelet Function ◽  
Whole Blood ◽  
Clinical Characteristics ◽  
Paroxysmal Nocturnal Haemoglobinuria ◽  
Clot Formation ◽  
Haematopoietic Stem Cell ◽  
Activation Markers ◽  
Platelet Surface ◽  
The Impact

Introduction Paroxysmal nocturnal haemoglobinuria (PNH) is a rare acquired clonal haematopoietic stem cell disorder characterised by haemolytic anaemia, thrombosis and bone marrow failure. Thrombosis is considered a major cause for high morbidity and mortality in PNH patients (Hillmen P et al N Eng J Med 1995). However, the underlying mechanisms of thrombosis in PNH are still poorly understood. Various factors including defective interactions between the complement system, platelets and coagulation systems have been proposed (Peacock-Young B et al Haematologica 2017). Platelet function and clot formation in this disorder have not been comprehensively evaluated. Here we describe standard and novel techniques to evaluate platelet function in blood from PNH patients, to elucidate the underlying pro-thrombotic mechanisms. Methods Whole blood was collected from five PNH patients and compared to same-day healthy donor (HD) controls. The samples were collected at trough for those patients who were on eculizumab (Complement C5 inhibitor used in the treatment of PNH). Surface levels of platelet adhesion proteins and activation markers P-selectin and phosphatidylserine (PS) were assessed using flow cytometry. Plasma soluble GPVI (a platelet-specific activation marker) and cytokine levels were measured by ELISA. Clot formation was assessed by viscoelastic testing (ROTEM). Adhesion of platelets to collagen under flow in a whole blood assay was evaluated by Digital Holographic Microscopy (DHM) and thrombus height, surface area and volume quantified using custom-built MatLab-based software. Results Clinical characteristics of PNH patients were highly variable: two patients had history of thrombosis, three patients were on eculizumab, four patients were thrombocytopenic (<150x109/L) and three were haemolysing. Platelet surface levels of adhesion/signalling receptor proteins including glycoproteins (GP) Iba, GPVI, aIIbb3, and ADAM10 (membrane expressed enzyme responsible for shedding GPVI from the surface) were all at lower ends of HD ranges. P-selectin and PS levels under resting and activated conditions and plasma soluble GPVI levels were comparable to same day HD. There were no differences between HD and PNH groups for levels of interleukin (IL) -6, IL-1β, tumour necrosis factor α, IL-17A, interferon γ or monocyte chemoattractant protein-1 (MCP-1). ROTEM analysis revealed slower formation of smaller clots in PNH patients, which correlated with their platelet count. Peak thrombus height and volume analysed by DHM were not different from data obtained with HD blood at both venous and arterial shear rates. However, both parameters were increased in PNH samples at arterial shearrate, when adjusted for platelet count (p<0.01). Conclusion Analysis of these PNH patients with highly variable clinical characteristics did not identify a unifying platelet lesion. DHM could detect and quantify parameters of small thrombi in real time, in PNH patient samples and these data were consistent with enhanced thrombogenic potential in PNH patients. Mechanisms beyond platelet activation that contribute to increased thrombosis in these patients and the impact of eculizumab therapy on thrombotic propensity need to be explored further. Disclosures D'Rozario: Alexion: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Platelet Function, Size and Yield in Whole Blood and in Platelet-Rich Plasma Prepared Using Differing Centrifugation Force and Time in Domestic and Food-Producing Animals

1983 ◽  
Vol 50 (04) ◽  
pp. 838-843 ◽  
Author(s):  
R M Clemmons ◽  
E L Bliss ◽  
M R Dorsey-Lee ◽  
C L Seachord ◽  
K M Meyers
Keyword(s):  
Platelet Aggregation ◽  
Whole Blood ◽  
Platelet Rich Plasma ◽  
Adenosine Diphosphate ◽  
Blood Platelet ◽  
Relative Difference ◽  
Sample Volume ◽  
Gravitational Forces ◽  
Induced Aggregation ◽  
Cell Volumes

SummaryThe effects of centrifugation force and time upon platelets function, mean platelet volume and platelet yield were compared with whole blood platelet counts and size in citrated blood samples from the bovine, canine, caprine, equine, feline, ovine and porcine species. The results were similar, for a given species, irregardless of sample volume. Bovine, caprine, feline and ovine platelet yields and mean platelet volumes were maximal when platelet-rich plasma was prepared using longer centrifugation times and lower gravitational forces. Canine, equine and porcine platelet yields and mean platelet volumes were maximal when platelet-rich plasma was prepared using shorter centrifugation times and higher gravitational forces. Platelet aggregation to adenosine diphosphate or arachidonic add was not effected by the method of platelet-rich plasma preparation in bovine, caprine, feline, ovine or pordne platelets. Equine platelet aggregation was maximal when platelet-rich plasma was prepared using longer centrifugation times and lower gravitational forces. Canine platelet aggregation, particularly arachidonic add-induced aggregation, was maximal when platelet-rich plasma was prepared using short centrifugation times and higher gravitational forces. It appeared that the effects of centrifugation parameters upon platelet yield depended upon the relative difference between platelet and red blood cell volumes.

Pre-analytical sampling and storage conditions of Amyloid-β peptides in venous and capillary blood

2020 ◽  
Author(s):  
Marlena Walter ◽  
Jens Wiltfang ◽  
Jonathan Vogelgsang
Keyword(s):  
Whole Blood ◽  
Room Temperature ◽  
Amyloid Β ◽  
Storage Conditions ◽  
Capillary Blood ◽  
Research Practice ◽  
Aβ Peptides ◽  
Standard Operating ◽  
The Impact ◽  
And Storage

Abstract BACKGROUND: We analyzed the impact of pre-analytical storage and sampling conditions on plasma Amyloid-β (Aβ) peptides.METHODS: Venous or capillary blood was collected and either stored as plasma or whole blood at room temperature or 4°C. Aβ 40 and Aβ 42 levels were measured using a chemiluminescence sandwich immunoassay.RESULTS: Aβ 40 , Aβ 42 and Aβ 42/40 levels significantly decreased during storage at room temperature in whole blood or plasma, starting at 6 hours after sampling. While Aβ 42 was most prone to the time of storage, Aβ 42/40 was stable up to 24 hours. Storing whole blood or plasma at 4°C led to stable Aβ peptide concentrations up to 72 hours. In addition, Aβ peptides could be measured in capillary blood. While Aβ 40 and Aβ 42 concentrations were slightly lower in capillary blood and started decreasing during storage conditions at 4°C, the Aβ 42/40 ratio remained constant up to 72 hours and was comparable to venous whole blood.CONCLUSION: These findings provide information that need to be respected in pre-analytical standard operating procedures for clinical and research practice to generate most reliable plasma Aβ measurements.

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