Sample Preparation as a Critical Aspect of Blood Platelet Mitochondrial Respiration Measurements—The Impact of Platelet Activation on Mitochondrial Respiration

Blood platelets are considered as promising candidates as easily-accessible biomarkers of mitochondrial functioning. However, their high sensitivity to various stimulus types may potentially affect mitochondrial respiration and lead to artefactual outcomes. Therefore, it is crucial to identify the factors associated with platelet preparation that may lead to changes in mitochondrial respiration. A combination of flow cytometry and advanced respirometry was used to examine the effect of blood anticoagulants, the media used to suspend isolated platelets, respiration buffers, storage time and ADP stimulation on platelet activation and platelet mitochondria respiration. Our results clearly show that all the mentioned factors can affect platelet mitochondrial respiration. Briefly, (i) the use of EDTA as anticoagulant led to a significant increase in the dissipative component of respiration (LEAK), (ii) the use of plasma for the suspension of isolated platelets with MiR05 as a respiration buffer allows high electron transfer capacity and low platelet activation, and (iii) ADP stimulation increases physiological coupling respiration (ROUTINE). Significant associations were observed between platelet activation markers and mitochondrial respiration at different preparation steps; however, the fact that these relationships were not always apparent suggests that the method of platelet preparation may have a greater impact on mitochondrial respiration than the platelet activation itself.

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Platelet PI3K Modulates Innate Leukocyte Extravasation during Acid-Induced Acute Lung Inflammation

Thrombosis and Haemostasis ◽

10.1055/s-0039-1693693 ◽

2019 ◽

Vol 119 (10) ◽

pp. 1642-1654 ◽

Cited By ~ 1

Author(s):

Julia Barbara Kral-Pointner ◽

Waltraud Cornelia Schrottmaier ◽

Manuel Salzmann ◽

Marion Mussbacher ◽

Georg Johannes Schmidt ◽

...

Keyword(s):

Platelet Function ◽

Lung Inflammation ◽

Blood Platelets ◽

Regulatory Subunit ◽

Interleukin 12 ◽

Pi3k Signaling ◽

Acute Lung Inflammation ◽

Activation Markers ◽

Leukocyte Extravasation ◽

The Impact

Introduction Blood platelets are increasingly recognized as modulators of leukocyte effector functions in various pathologies including acute lung injury (ALI). ALI is a life-threatening disease, caused by damage to the alveolar epi- and endothelium. Excessive accumulation of leukocytes leads to severe lung inflammation, resulting in impaired lung function and hypoxemia. Objective Since leukocyte migration is modulated by activated platelets and phosphatidylinositol 3-kinase (PI3K) signaling is involved in platelet function, we aimed to elucidate the effect of PI3K on platelet-mediated immune responses. Materials and Methods We generated a mouse model with a platelet-specific deletion of p85α, the most important regulatory subunit of the class IA PI3K, and evaluated platelet function and platelet–leukocyte interactions. Moreover, we analyzed the impact of platelet-specific p85α gene deficiency during sterile peritonitis and acid-induced ALI. Results In vitro analyses of platelets revealed that lack of p85α led to decreased downstream signaling and diminished expression of surface activation markers, for example, CD62P and CD63, as well as reduced platelet aggregation. Moreover, platelet PI3K essentially mediated direct interactions of platelets with monocytes and neutrophils. In mice, platelet-specific p85α deficiency prevented leukocyte infiltration into the peritoneum and the bronchoalveolar compartment during sterile peritonitis and ALI, respectively. Additionally, the release of the inflammatory cytokine interleukin-12/23 was diminished in platelet p85α-deficient mice during ALI. In contrast to PI3K, neither overexpression nor depletion of platelet phosphatase and tensin homolog, the endogenous antagonist of PI3K, significantly modulated platelet function. Conclusion Our data indicate a crucial role of platelet PI3K signaling for leukocyte extravasation upon inflammatory stimuli in various diseases models.

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Increased Pro-Thrombotic Platelet Activity Associated with Thrombin/PAR1-Dependent Pathway Disorder in Patients with Secondary Progressive Multiple Sclerosis

International Journal of Molecular Sciences ◽

10.3390/ijms21207722 ◽

2020 ◽

Vol 21 (20) ◽

pp. 7722

Author(s):

Angela Dziedzic ◽

Elzbieta Miller ◽

Michal Bijak ◽

Lukasz Przyslo ◽

Joanna Saluk-Bijak

Keyword(s):

Multiple Sclerosis ◽

Mrna Expression ◽

Platelet Activation ◽

Blood Platelets ◽

Surface Expression ◽

Epidemiological Studies ◽

Progressive Multiple Sclerosis ◽

Apolipoprotein A1 ◽

Platelet Activity ◽

Activation Markers

Epidemiological studies confirm the high risk of ischemic events in multiple sclerosis (MS) that are associated with increased pro-thrombotic activity of blood platelets. The most potent physiological platelet agonist is thrombin, which activates platelets via cleavage of specific protease-activated receptors (PARs). Our current study is aimed to determine the potential genetics and proteomic abnormalities of PAR1 in both platelets and megakaryocytes, which may have thromboembolic consequences in the course of MS. The obtained results were correlated with the expression level of platelet and megakaryocyte transcripts for APOA1 and A2M genes encoding atherosclerosis biomarkers: apolipoprotein A1 (ApoA1) and α-2-macroglobulin (α2M), respectively. Moreover, PAR1 functionality in MS platelets was assessed by flow cytometry, determining the level of platelet–platelet and platelet–leukocyte aggregates, platelet microparticles and surface expression of P-selectin. As a PAR1 agonist, the synthetic TRAP-6 peptide was used, which made it possible to achieve platelet activation in whole blood without triggering clotting. Comparative analyses showed an elevated level of platelet activation markers in the blood of MS patients compared to controls. The mRNA expression of gene coding α2M was upregulated, whilst ApoA1 was down-regulated, both in platelets and megakaryocytes from MS patients. Furthermore, we observed an increase in both mRNA expression and surface density of PAR1 in platelets and megakaryocytes in MS compared to controls. Both the level of platelet activation markers and PAR1 expression showed a high correlation with the expression of transcripts for APOA1 and A2M genes.

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The control of blood platelets by cAMP signalling

Biochemical Society Transactions ◽

10.1042/bst20130278 ◽

2014 ◽

Vol 42 (2) ◽

pp. 289-294 ◽

Cited By ~ 13

Author(s):

Zaher Raslan ◽

Khalid M. Naseem

Keyword(s):

Protein Kinase ◽

Protein Kinase A ◽

Platelet Activation ◽

Blood Platelets ◽

Blood Platelet ◽

Platelet Inhibition ◽

Signalling Pathway ◽

Current Understanding ◽

Camp Signalling ◽

Kinase A

Blood platelet activation must be tightly regulated to ensure a balance between haemostasis and thrombosis. The cAMP signalling pathway is the most powerful endogenous regulator of blood platelet activation. PKA (protein kinase A), the foremost effector of cAMP signalling in platelets, phosphorylates a number of proteins that are thought to modulate multiple aspects of platelet activation. In the present mini-review, we outline our current understanding of cAMP-mediated platelet inhibition and discuss some of the issues that require clarification.

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The Changes of Blood Platelet Reactivity in the Presence of Crude Extracts Isolated from Leaves and Twigs of Elaeagnus Rhamnoides (L.) A. Nelson Measuring in Whole Blood

10.21203/rs.3.rs-1171071/v1 ◽

2021 ◽

Author(s):

Bartosz Skalski ◽

Joanna Rywaniak ◽

Jerzy Żuchowski ◽

Anna Stochmal ◽

Beata Olas

Keyword(s):

Phenolic Compounds ◽

Platelet Activation ◽

Whole Blood ◽

Leaf Extract ◽

Platelet Reactivity ◽

Blood Platelets ◽

Blood Platelet ◽

Surface Expression ◽

Sea Buckthorn ◽

Crude Extracts

Abstract Uncontrolled blood platelet activation is an important risk factor of cardiovascular disease (CVDs). Various studies on phenolic compounds indicate that they have a protective effect on the cardiovascular system through different mechanisms, including the reduction of blood platelet activation. One of the plants that is particularly rich in phenolic compounds is sea buckthorn (Elaeagnus rhamnoides (L.) A. Nelson). The aim of the present study in vitro was to determine the anti-platelet properties of crude extracts isolated from leaves and twigs of E. rhamnoides (L.) A. Nelson in whole blood using flow cytometric and total thrombus-formation analysis system (T-TAS). The aim of our study was also analyze of blood platelet proteoms in the presence of different sea buckthorn extracts. A significant new finding is a decrease surface expression of P-selectin on blood platelets stimulated by 10 µM ADP and 10 µg/mL collagen, and a decrease surface expression of GPIIb/IIIa active complex on non-activated platelets and platelets stimulated by 10 µM ADP and 10 µg/mL collagen in the presence of sea buckthorn leaf extract (especially at the concentration 50 µg/mL). The twig extract also displayed antiplatelet potential. However, this activity was higher in the leaf extract than in the twig extract in whole blood. In addition, our present findings clearly demonstrate that investigated plant extracts have anticoagulant properties (measured by T-TAS). Therefore, the two tested extracts may be promising candidates for the natural anti-platelet and anticoagulant supplements.

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Effect of ultrapure lipopolysaccharides derived from diverse bacterial species on the modulation of platelet activation

Scientific Reports ◽

10.1038/s41598-019-54617-w ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 4

Author(s):

Thomas M. Vallance ◽

Divyashree Ravishankar ◽

Dina A. I. Albadawi ◽

Harry Layfield ◽

Jonathan Sheard ◽

...

Keyword(s):

Platelet Activation ◽

Platelet Reactivity ◽

Platelet Rich Plasma ◽

Bacterial Species ◽

Human Platelets ◽

Innate Immune Responses ◽

Experimental Conditions ◽

Activation Markers ◽

The Impact ◽

Inside Out

AbstractPlatelets are small circulating blood cells that play essential roles in the maintenance of haemostasis via blood clotting. However, they also play critical roles in the regulation of innate immune responses. Inflammatory receptors, specifically Toll-like receptor (TLR)-4, have been reported to modify platelet reactivity. A plethora of studies have reported controversial functions of TLR4 in the modulation of platelet function using various chemotypes and preparations of its ligand, lipopolysaccharide (LPS). The method of preparation of LPS may explain these discrepancies however this is not fully understood. Hence, to determine the impact of LPS on platelet activation, we used ultrapure preparations of LPS from Escherichia coli (LPSEC), Salmonella minnesota (LPSSM), and Rhodobacter sphaeroides (LPSRS) and examined their actions under diverse experimental conditions in human platelets. LPSEC did not affect platelet activation markers such as inside-out signalling to integrin αIIbβ3 or P-selectin exposure upon agonist-induced activation in platelet-rich plasma or whole blood whereas LPSSM and LPSRS inhibited platelet activation under specific conditions at supraphysiological concentrations. Overall, our data demonstrate that platelet activation is not largely influenced by any of the ultrapure LPS chemotypes used in this study on their own except under certain conditions.

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Platelet Counts and Platelet Activation Markers in Obese Subjects

Mediators of Inflammation ◽

10.1155/2008/834153 ◽

2008 ◽

Vol 2008 ◽

pp. 1-6 ◽

Cited By ~ 44

Author(s):

Dorit Samocha-Bonet ◽

Dan Justo ◽

Ori Rogowski ◽

Nili Saar ◽

Subchi Abu-Abeid ◽

...

Keyword(s):

Flow Cytometry ◽

Platelet Activation ◽

Annexin V ◽

High Sensitivity ◽

Serum Levels ◽

Normal Weight ◽

Morbidly Obese ◽

Activation Markers ◽

Platelet Counts ◽

Hs Crp

Objective. In this work we studied the correlation between platelet count, platelet activation, and systemic inflammation in overweight, obese, and morbidly obese individuals.Methods and subjects. A total of 6319 individuals participated in the study. Complete blood counts, high sensitivity C-reactive protein (hs-CRP) serum levels, and body mass index (BMI) were measured during routine checkups. Platelet activation markers were studied among 30 obese (BMI = 41 8 kg/m2) and 35 nonobese (BMI = 24 3 kg/m2) individuals. Platelet activation status was evaluated by flow cytometry using specific antibodies against the activated platelet membrane glycoprotein IIb/IIIa, p-selectin (CD-62 p), and binding of Annexin-V to platelet anionic phospholipids.Results. Overweight, obese, and morbidly obese females had significantly elevated platelet counts () compared with normal-weight females. No significant elevation of platelet counts was observed in the male subgroups. A significant age adjusted correlation between BMI and platelet counts () was found among females. This correlation was attenuated () after adjustment for hs-CRP concentrations. The flow cytometry analysis of platelets showed no significant differences in activation marker expression between nonobese and obese individuals.Discussion. Obesity may be associated with elevated platelet counts in females with chronic inflammation. Obesity is not associated with increased platelet activation.

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Inflammation and Platelet Activation After COVID-19 Vaccines - Possible Mechanisms Behind Vaccine-Induced Immune Thrombocytopenia and Thrombosis

Frontiers in Immunology ◽

10.3389/fimmu.2021.779453 ◽

2021 ◽

Vol 12 ◽

Author(s):

Sisse R. Ostrowski ◽

Ole S. Søgaard ◽

Martin Tolstrup ◽

Nina B. Stærke ◽

Jens Lundgren ◽

...

Keyword(s):

Platelet Activation ◽

Immune Thrombocytopenia ◽

Thrombin Generation ◽

High Titer ◽

Adenovirus Vector ◽

Activation Markers ◽

Platelet Factor ◽

Pronounced Increase ◽

Impedance Aggregometry ◽

The Impact

Introduction of vaccines against COVID-19 has provided the most promising chance to control the world-wide COVID-19 pandemic. However, the adenovirus-vector based Oxford/AstraZeneca [ChAdOx1] (AZ) and Johnson & Johnson [Ad26.CoV2.S] COVID-19 vaccines have been linked with serious thromboembolic events combined with thrombocytopenia, denominated Vaccine-induced Immune Thrombocytopenia and Thrombosis (VITT). The pathogenesis of COVID-19 VITT remain incompletely understood; especially the initial events that trigger platelet activation, platelet factor (PF)4 release, complex formation and PF4 antibody production are puzzling. This is a prospective study investigating the impact of different COVID-19 vaccines on inflammation (CRP, TNF-α, IL-1β, IL-6, IL-8, IL-10), vascular endothelial activation (syndecan-1, thrombomodulin, E-selectin, ICAM-1, ICAM-3, VCAM-1), platelet activation (P-selectin, TGF-β, sCD40L) and aggregation (Multiplate® impedance aggregometry), whole blood coagulation (ROTEM®), thrombin generation and PF4 antibodies to reveal potential differences between AZ and mRNA vaccines in individuals without VITT. The study included 80 (55 AZ and 55 mRNA) vaccinated individuals and 55 non-vaccinated age- and gender matched healthy controls. The main findings where that both vaccines enhanced inflammation and platelet activation, though AZ vaccination induced a more pronounced increase in several inflammatory and platelet activation markers compared to mRNA vaccination and that post-vaccination thrombin generation was higher following AZ vaccination compared to mRNA vaccination. No difference in neither the PF4 antibody level nor the proportion of individuals with positive PF4 antibodies were observed between the vaccine groups. This is the first study to report enhanced inflammation, platelet activation and thrombin generation following AZ vaccination compared to mRNA vaccination in a head-to-head comparison. We speculate that specific components of the AZ adenovirus vector may serve as initial trigger(s) of (hyper)inflammation, platelet activation and thrombin generation, potentially lowering the threshold for a cascade of events that both trigger complications related to excessive inflammation, platelet and coagulation activation as observed in epidemiological studies and promote development of VITT when combined with high-titer functionally active PF4 antibodies.

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Preparations from Selected Cucurbit Vegetables as Antiplatelet Agents – An in Vitro Study

10.21203/rs.3.rs-923749/v1 ◽

2021 ◽

Author(s):

Agata Rolnik ◽

Bartosz Skalski ◽

Anna Stochmal ◽

Beata Olas

Keyword(s):

Platelet Activation ◽

Cucurbita Pepo ◽

Thrombus Formation ◽

Blood Platelets ◽

In Vitro Study ◽

Blood Platelet ◽

Arachidonic Acid Metabolism ◽

Platelet Activity ◽

Activated Platelets

Abstract Increased blood platelet activation plays an important role in cardiovascular diseases (CVDs). Recent experiments indicate that certain fruits and vegetables, including onion, garlic, and beetroot, have anti-platelet potential and therefore may reduce the likelihood of CVDs. While vegetables from the Cucuritaceae family are known to exerting beneficial antioxidant and anti-inflammatory effects, their effects on blood platelet activation are poorly understood. Therefore, the aim of the present study was to determine the effect on platelet adhesion of preparations from selected cucurbits: pumpkin (Cucirbita pepo; fruit without seeds), zucchini (Cucurbita pepo convar. giromontina; fruit with seeds), cucumber (Cucumis sativus; fruit with seeds), white pattypan squash (Cucurbita pepo var. patisoniana; fruit without seeds) and yellow pattypan squash (Cucurbita pepo var. patisoniana, fruit without seeds). It also evaluates the activity of these preparations on enzymatic lipid peroxidation in thrombin-activated washed blood platelets by TBARS assay. The study also determines the anti-platelet and anticoagulant properties of these five cucurbit preparations in whole blood by flow cytometry and with the total thrombus-formation analysis system (T-TAS) and evaluates the cytotoxicity of the tested preparations against platelets based on LDH activity. The results indicate that the yellow Cucurbita pepo var. patisoniana preparation demonstrated stronger anti-platelet properties than the other tested preparations, reducing the adhesion of thrombin-activated platelets to collagen/fibrinogen, and inhibiting arachidonic acid metabolism and GPIIb/IIIa expression on 10 µM ADP-activated platelets. None of the preparations was found to cause platelet lysis. Our findings provide new information on the anti-platelet activity of the tested cucurbit preparations and their potential for treating CVDs associated with platelet hyperactivity.

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Optimum Design of a Transportation Scheme for Healthcare Supply Chain Management: The Effect of Energy Consumption

Energies ◽

10.3390/en12142789 ◽

2019 ◽

Vol 12 (14) ◽

pp. 2789 ◽

Cited By ~ 8

Author(s):

Jihed Jemai ◽

Biswajit Sarkar

Keyword(s):

Supply Chain ◽

Energy Consumption ◽

Carbon Emissions ◽

Blood Platelets ◽

Blood Platelet ◽

Total Cost ◽

Location Allocation ◽

Numerical Studies ◽

Proposed Model ◽

The Impact

The perishability of blood platelets complicates the management of their supply chain. This paper studies the impact of energy consumption and carbon emissions of transportation activities in a blood platelet supply chain. Energy consumption and carbon emissions vary significantly, and the effective location-allocation of blood facilities is a key strategy for the optimal use of energy. The total cost of the supply chain for perishable products is minimized when energy consumption is optimized. The proposed model is too complex to be solved with existing methodologies; therefore, mathematical tools are used to solve it. A numerical experiment is carried out to validate the proposed model, and graphical representations are presented for better visualization of the study’s outcomes. The results of the numerical studies confirm that the selected locations of blood facilities are optimal for the maximization of energy efficiency and minimization of the total cost.

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Preparations from selected cucurbit vegetables as antiplatelet agents

Scientific Reports ◽

10.1038/s41598-021-02235-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Agata Rolnik ◽

Bartosz Skalski ◽

Anna Stochmal ◽

Beata Olas

Keyword(s):

Platelet Activation ◽

Cucurbita Pepo ◽

Fruits And Vegetables ◽

Thrombus Formation ◽

Blood Platelets ◽

Antiplatelet Agents ◽

Blood Platelet ◽

Arachidonic Acid Metabolism ◽

Platelet Activity ◽

Activated Platelets

AbstractIncreased blood platelet activation plays an important role in cardiovascular diseases (CVDs). Recent experiments indicate that certain fruits and vegetables, including onion, garlic, and beetroot, have anti-platelet potential and therefore may reduce the likelihood of CVDs. While vegetables from the Cucuritaceae family are known to exerting beneficial antioxidant and anti-inflammatory effects, their effects on blood platelet activation are poorly understood. Therefore, the aim of the present study was to determine the effect on platelet adhesion of preparations from selected cucurbits: pumpkin (Cucurbita pepo; fruit without seeds), zucchini (Cucurbita pepo convar. giromontina; fruit with seeds), cucumber (Cucumis sativus; fruit with seeds), white pattypan squash (Cucurbita pepo var. patisoniana; fruit without seeds) and yellow pattypan squash (Cucurbita pepo var. patisoniana, fruit without seeds). It also evaluates the activity of these preparations on enzymatic lipid peroxidation in thrombin-activated washed blood platelets by TBARS assay. The study also determines the anti-platelet properties of these five cucurbit preparations in whole blood by flow cytometry and with the total thrombus-formation analysis system (T-TAS) and evaluates the cytotoxicity of the tested preparations against platelets based on LDH activity. The results indicate that the yellow Cucurbita pepo var. patisoniana preparation demonstrated stronger anti-platelet properties than the other tested preparations, reducing the adhesion of thrombin-activated platelets to collagen/fibrinogen, and inhibiting arachidonic acid metabolism and GPIIb/IIIa expression on 10 µM ADP-activated platelets. None of the preparations was found to cause platelet lysis. Our findings provide new information on the anti-platelet activity of the tested cucurbit preparations and their potential for treating CVDs associated with platelet hyperactivity.

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