Mitochondrial Respiration of Platelets: Comparison of Isolation Methods

Multiple non-aggregatory functions of human platelets (PLT) are widely acknowledged, yet their functional examination is limited mainly due to a lack of standardized isolation and analytic methods. Platelet apheresis (PA) is an established clinical method for PLT isolation aiming at the treatment of bleeding diathesis in severe thrombocytopenia. On the other hand, density gradient centrifugation (DC) is an isolation method applied in research for the analysis of the mitochondrial metabolic profile of oxidative phosphorylation (OXPHOS) in PLT obtained from small samples of human blood. We studied PLT obtained from 29 healthy donors by high-resolution respirometry for comparison of PA and DC isolates. ROUTINE respiration and electron transfer capacity of living PLT isolated by PA were significantly higher than in the DC group, whereas plasma membrane permeabilization resulted in a 57% decrease of succinate oxidation in PA compared to DC. These differences were eliminated after washing the PA platelets with phosphate buffer containing 10 mmol·L−1 ethylene glycol-bis (2-aminoethyl ether)-N,N,N′,N′-tetra-acetic acid, suggesting that several components, particularly Ca2+ and fuel substrates, were carried over into the respiratory assay from the serum in PA. A simple washing step was sufficient to enable functional mitochondrial analysis in subsamples obtained from PA. The combination of the standard clinical PA isolation procedure with PLT quality control and routine mitochondrial OXPHOS diagnostics meets an acute clinical demand in biomedical research of patients suffering from thrombocytopenia and metabolic diseases.

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The Effect Of Prostacyclin (PGI2) On The Procoagulant Activity Of Human Platelets

10.1055/s-0038-1652806 ◽

1981 ◽

Author(s):

J Brox ◽

B Østerud

Keyword(s):

Platelet Aggregation ◽

Gradient Centrifugation ◽

Inhibitory Effect ◽

Human Platelets ◽

Serotonin Release ◽

Procoagulant Activity ◽

Factor V ◽

Platelet Factor ◽

Healthy Donors

Platelets from healthy donors were isolated by albumin- gradient centrifugation and gelfiltration. The platelets were exposed to thrombin, collagen, and ADP separately, and thrombin and collagen in combination. The concentrations used were the lowest that gave maximal aggregation. The following parameters were assayed: aggregation, platelet factor 3(PF 3), Factor V-Va(F.V-Va), total procoagulant activity (TPA, which measures the combined activity of PF 3 and F.V-Va), and serotonin release. The effect of various concentrations of PGI2 on these parameters was examined.Thrombin was more potent than collagen, and collagen was more potent than ADP in stimulating the procoagulant activity and serotonin release (i.e. thrombin generated 33%, collagen 14%, ADP 3% TPA as compared to 100% for lysed platelets). Thrombin alone was equally strong as thrombin and collagen in combination in regard to TPA. The platelet aggregation was maximal in all these experiments.PGI2 (1.4x10-8M) inhibited very efficiently aggregation, serotonin release, TPA, PF 3 and F.V-Va activity when the platelets were stimulated with thrombin, collagen or ADP. When platelets were exposed to thrombin and collagen simul- tanously, the inhibitory effect of PGI2 on TPA decreased. PGI2 concentration of 1.4xlO-7M in such platelet mixtures inhibited TPA by 30-40% whereas the same PGI2 dose inhibited TPA by 80-90% when thrombin and collagen were used separately. In these experiments platelet aggregation was less than 20%.This study demonstrates that PGI2 strongly inhibits the availability of the platelet procoagulant activity, and PGI2 may therefore also slow down the generation of thrombin.

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Antithrombin III Antigen in Human Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1660078 ◽

1985 ◽

Vol 54 (03) ◽

pp. 599-602 ◽

Cited By ~ 2

Author(s):

M Léon Alhenc-Gelas ◽

M Aiach ◽

A Gorenflot ◽

J P Andreux

Keyword(s):

Arachidonic Acid ◽

Enzyme Immunoassay ◽

Antithrombin Iii ◽

Human Platelets ◽

Mean Value ◽

Normal Blood ◽

Freezing And Thawing ◽

Healthy Donors ◽

At Iii ◽

Storage Granules

SummaryImmunoreactive AT III was found in human platelets. AT III antigen was quantified in platelets taken from each of 17 healthy donors by a specific competitive enzyme immunoassay using purified AT III and AT III antibodies. AT III antigen levels in extracts of washed platelets disrupted by freezing and thawing ranged from 32 to 140 ng per 109 platelets with a mean value of 70.3 ± 27.3. When stimulated by arachidonic acid, the platelets released AT III antigen together with immunoreactive fibrinogen. These results show that AT III is present in platelets at a level corresponding to approximately 0.01% of total antithrombin in normal blood, and suggest that platelet AT III, like fibrinogen, is contained in the storage granules.

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Quantitative and Qualitative Analysis of Regulatory T Cells (Tregs) in B Cell Chronic Lymphocytic Leukemia (B-CLL)

Blood ◽

10.1182/blood.v126.23.2928.2928 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2928-2928

Author(s):

Vassiliki Mpakou ◽

Dikea-Eleni Ioannidou ◽

Myrofora Vikentiou ◽

Eugenia Konsta ◽

Aris Spathis ◽

...

Keyword(s):

Functional Analysis ◽

Mononuclear Cells ◽

Gradient Centrifugation ◽

Annexin V ◽

Treg Cells ◽

Effector Cells ◽

T Effector Cells ◽

Healthy Donors ◽

Brdu Assay

Abstract Introduction: T regulatory cells are immunosuppressive cells considered to play an important role in cancer biology and autoimmunity by suppressing host immune response and autoreactive lymphocytes respectively. Several studies reveal that Treg cells act by suppressing anti-tumor immune response, through the targeting of other immune cells, such as T cells, B cells and dendritic cells. Accumulated data indicate a significant role of T cell dysfunction in the pathogenesis of CLL. Aims: The scope of this study is the analysis of numerical and functional abnormalities of Tregs in B-CLL with the view to elucidate their role in the pathogenesis of the disease. Methods: Treg cells derived from 44 untreated B-CLL patients with a median age 62 and 17 healthy donors were analyzed by Flow cytometry, using the following antibodies: CD45Ro-FITC/CD45RA-PE/CD4-ECD/CD25-PC5/CD127-PC7, CD1a-FITC/CD137-PE/CD4-ECD/CD25-PC5/CD127-PC7, CD95-FITC/cyCD152-PE/CD4-ECD/CD25-PC5/CD127-PC7, beads/FoxP3-PE/CD4-ECD/CD25-PC5/CD127-PC7, Annexin V-FITC/CD4-ECD/CD25-PC5/CD127-PC7. For the functional analysis, peripheral blood was obtained from 20 patients with B-CLL. Mononuclear cells were isolated using Ficoll-Paque gradient centrifugation. CD4+ CD25+ (Treg cells), CD4+ CD25- (T effectοr cells, Teff), CD5+ CD19+ (B-CLL) and CD5- CD19+ (Normal B, NB) cells were separated using magnetic antibody cell sorting. To test the functionality of the assayed Tregs, the isolated cell populations were cultured in a 96-well plate (Tregs, Teff, B-CLL cells, NB cells, B-CLL cells: Tregs in 1:20 ratio, B-CLL cells: Teff in 1:20 ratio, NB cells: Tregs in 1:20 ratio, NB cells: Teff in 1:20 ratio) and their proliferative capacity was measured using the BrdU assay. To further analyze the functional role of Tregs, peripheral blood was obtained from 22 patients with CLL and 22 healthy donors. Mononuclear cells were isolated using Ficoll-Paque gradient centrifugation. CD4+ CD25+ CD127dim/- (Treg cells), CD5+ CD19+ (B-CLL) and CD5- CD19+ (Normal B, NB) cells were separated using magnetic antibody cell sorting and were co-cultured in a 96-well plate in a 1:10 ratio. The apoptosis of B cells was determined by the Annexin V/PI method. Results: FACS analysis of the Treg cells resulted at the following observations: The Treg absolute cell number (cells/μL), estimated either as the number of CD4+ CD25+ CD127- cells or as the number of CD4+ CD25+ FoxP3+ cells, was statistically significantly higher in patients' samples than in controls (CD127- 21.65 vs 7.35, p=0.001; FoxP3+ 20.42 vs 6.5, p= 0.001). Annexin V expression in Treg cells from BCLL patients was significantly lower compared to controls (3.626 vs 38.615, p=0.003). The functional analysis of Treg cells through BrdU assay indicated that CLL Tregs were able to suppress the proliferation of Teff cells (p=0.002) and that Teff cells were in turn able to significantly suppress the proliferation of B-CLL cells (p=0.05). Moreover, FACS analysis through Annexin V/PI method indicated that Treg CLL cells significantly decrease the apoptosis rate of NB cells after their co-culturing, compared to NB cells (p<0,02). On the contrary, healthy donors derived Treg cells significantly increase the apoptosis of B-CLL cells after their co-culturing, compared to B-CLL cells (p<0.025). Interestingly, no significant alterations were observed after culturing NB cells with Tregs from healthy donors and B-CLL cells with Treg CLL cells. Conclusions: In CLL patients, Treg cells are significantly higher and present with lower apoptotic levels compared to healthy donors. The functional analysis indicates that T effector cells suppress the proliferation of B-CLL cells and T effector cells are suppressed by Tregs indicating that the increased number of Tregs observed in CLL contributes indirectly to the proliferation of the CLL clone. These data are further supported by our observations that CLL derived Treg cells appear rather incapable of inducing apoptosis of both NB cells and B-CLL cells, in contrast to normal Tregs, suggesting an immunoediting effect of B-CLL cells on Tregs which negatively affects the functionality of the latter. Therefore, Treg cells in CLL do not efficiently eliminate the abnormal clone and play an important role in the pathogenesis of the disease. The molecular underlying mechanisms need to be further elucidated. Disclosures No relevant conflicts of interest to declare.

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Anticoagulant and antithrombotic properties of platelet protease nexin-1

Blood ◽

10.1182/blood-2009-04-217240 ◽

2010 ◽

Vol 115 (1) ◽

pp. 97-106 ◽

Cited By ~ 45

Author(s):

Yacine Boulaftali ◽

Frédéric Adam ◽

Laurence Venisse ◽

Véronique Ollivier ◽

Benjamin Richard ◽

...

Keyword(s):

Ex Vivo ◽

Thrombus Formation ◽

Human Platelets ◽

Platelet Surface ◽

Type Plasminogen Activator ◽

Healthy Donors ◽

Protease Nexin ◽

Deficient Mice

AbstractProtease nexin–1 (PN-1) is a serpin that inhibits plasminogen activators, plasmin, and thrombin. PN-1 is barely detectable in plasma but is expressed by platelets. Here, we studied platelet PN-1 in resting and activated conditions and its function in thrombosis. Studies on human platelets from healthy donors and from patients with a Gray platelet syndrome demonstrate that PN-1 is present both at the platelet surface and in α-granules. The role of PN-1 was investigated in vitro using human platelets incubated with a blocking antibody and using platelets from PN-1–deficient mice. Both approaches indicate that platelet PN-1 is active on thrombin and urokinase-type plasminogen activator. Blockade and deficiency of platelet PN-1 result in accelerated and increased tissue factor-induced thrombin generation as indicated by calibrated automated thrombography. Moreover, platelets from PN-1–deficient mice respond to subthreshold doses of thrombin, as assessed by P-selectin expression and platelet aggregation. Thrombus formation, induced ex vivo by collagen in blood flow conditions and in vivo by FeCl3-induced injury, is significantly increased in PN-1–deficient mice, demonstrating the antithrombotic properties of platelet PN-1. Platelet PN-1 is thus a key player in the thrombotic process, whose negative regulatory role has been, up to now, markedly underestimated.

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Total parasite biomass but not peripheral parasitaemia is associated with endothelial and haematological perturbations in Plasmodium vivax patients

eLife ◽

10.7554/elife.71351 ◽

2021 ◽

Vol 10 ◽

Author(s):

João Luiz Silva-Filho ◽

João CK Dos-Santos ◽

Carla C Judice ◽

Dario Beraldi ◽

Kannan Venugopal ◽

...

Keyword(s):

Plasmodium Vivax ◽

Vivax Malaria ◽

Ex Vivo ◽

Parasite Burden ◽

Host Responses ◽

Severe Thrombocytopenia ◽

High Association ◽

Healthy Donors ◽

Circulating Markers ◽

Patient Subgroups

Plasmodium vivax is the major cause of human malaria in the Americas. How P. vivax infection can lead to poor clinical outcomes, despite low peripheral parasitaemia remains a matter of intense debate. Estimation of total P. vivax biomass based on circulating markers indicates existence of a predominant parasite population outside of circulation. In this study we investigate associations between both peripheral and total parasite biomass and host response in vivax malaria. We analysed parasite and host signatures in a cohort of uncomplicated vivax malaria patients from Manaus, Brazil, combining clinical and parasite parameters, multiplexed analysis of host responses and ex vivo assays. Patterns of clinical features, parasite burden and host signatures measured in plasma across the patient cohort were highly heterogenous. Further data deconvolution revealed two patient clusters, here termed Vivaxlow and Vivaxhigh. These patient subgroups were defined based on differences in total parasite biomass but not peripheral parasitaemia. Overall Vivaxlow patients clustered with healthy donors and Vivaxhigh patients showed more profound alterations in haematological parameters, endothelial cell (EC) activation and glycocalyx breakdown and levels of cytokines regulating different haematopoiesis pathways compared to Vivaxlow. Vivaxhigh patients presented more severe thrombocytopenia and lymphopenia, along with enrichment of neutrophils in the peripheral blood and increased neutrophil-to-lymphocyte ratio (NLCR). When patients' signatures were combined, high association of total parasite biomass with a subset of markers of EC activation, thrombocytopenia and lymphopenia severity was observed. Finally, machine learning models defined a combination of host parameters measured in the circulation that could predict the extent of parasite infection outside of circulation. Altogether, our data show that total parasite biomass is a better predictor of perturbations in host homeostasis in P. vivax patients than peripheral parasitaemia. This supports the emerging paradigm of a P. vivax tissue reservoir, in particular in the hematopoietic niche of bone marrow and spleen.

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The effect of thrombin on the density distribution of blood platelets: detection of activated platelets in the circulation

Blood ◽

10.1182/blood.v62.2.433.bloodjournal622433 ◽

1983 ◽

Vol 62 (2) ◽

pp. 433-438

Author(s):

B van Oost ◽

IH van Hien-Hagg ◽

AP Timmermans ◽

JJ Sixma

Keyword(s):

Cardiopulmonary Bypass ◽

Density Distribution ◽

Density Gradient Centrifugation ◽

Blood Platelets ◽

Gradient Centrifugation ◽

Buoyant Density ◽

Human Platelets ◽

Activated Platelets

The buoyant density of human platelets is decreased after they have been aggregated and induced to secrete their granule content by thrombin. This change in density was detected by discontinuous density gradient centrifugation using arabinogalactan (Stractan) solutions. The density decrease was dependent on the thrombin concentration and paralleled the extent of serotonin and beta-thromboglobulin secretion. The degranulated platelets maintained their integrity, and many of their functional properties. Mixtures of degranulated platelets and normal platelets could be resolved by Stractan gradient centrifugation and the number of degranulated platelets quantitated. Using this method, increased levels of less dense platelets were shown to occur after cardiopulmonary bypass. Assay of changes in platelet density by Stractan gradient centrifugation is a useful method for detection of activated platelets in vitro and in vivo.

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The Differentiation of Acid Phosphatases of Human Blood Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653683 ◽

1971 ◽

Vol 26 (02) ◽

pp. 353-361 ◽

Cited By ~ 8

Author(s):

H. D Kaulen ◽

R Gross

Keyword(s):

Rat Liver ◽

Blood Platelets ◽

Gradient Centrifugation ◽

Sucrose Density Gradient ◽

Human Platelets ◽

Acid Phosphatases ◽

Sucrose Density Gradient Centrifugation ◽

Lysosomal Fraction ◽

Hydrolysis Of ◽

Human Blood Platelets

SummaryThe Acid Phosphatase was tested in human platelets and in the rat liver mitochondrial-lysosomal fraction with p-nitrophenylphosphate and β-glycerophosphate as substrates. In the platelets the following differences were found between the hydrolysis of these two substrates, whereas in rat liver no such differences were observed. 1. The relative rates of hydrolysis and the pH optima for both substrates are different (pH 4.6 for the β-glycerophosphatase, pH 6.0 for the p-nitrophenylphosphatase in the platelets). 2. The p-nitrophenylphosphatase of the platelets is inhibited by p-chlormercuribenzoate and N-ethylmaleimide, but not by fluoride or L + tartrate, whereas the contrary is true for the platelet β-glycerophosphatase and the rat liver activities. 3. The platelet p-nitrophenylphosphatase is rapidly inactivated by preincubation at 40-45° C for 15 min, the other phosphatases are much more heat-resistant. 4. Sucrose density gradient centrifugation of platelet homgenates showed a separation of the two platelet phosphatase activities, the p-nitrophenylphosphatase with its maximum at lower densities than the β-glycerophosphatase.It is concluded that in human platelets there are at least two different Acid Phosphatases. The β-glycerophosphatase probably represents the lysosomal (as compared to the rat liver enzyme) phosphatase whereas the p-nitrophenylphosphatase of the platelets is a different enzyme whose subcellular localization and functions are as yet unknown.

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Determination of the Density Distribution of Human Platelets - Methodological Aspects and Comparison with Other Tests for Platelet Activation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657177 ◽

1982 ◽

Vol 47 (03) ◽

pp. 239-243 ◽

Cited By ~ 9

Author(s):

B A van Oost ◽

I H van Hien-Hagg ◽

B F E Veldhuyzen ◽

A P M Timmermans ◽

J J Sixma

Keyword(s):

Platelet Activation ◽

Gradient Centrifugation ◽

Buoyant Density ◽

Human Platelets ◽

Thrombotic Disease ◽

Platelet Aggregate ◽

Thrombotic Complications ◽

Methodological Aspects ◽

Function Testing

SummaryStimulation of human platelets to release results in decreased buoyant density. This decreased density provides a tool to detect circulating platelets which have participated in a thrombotic process. Platelet density gradient centrifugation using Stractan was standardized and the effects of anticoagulation, temperature, and osmolarity were investigated. In 7 out of 32 patients with thrombotic disease less dense platelets were found. Platelet activation in the patient group was also indicated by spontaneous aggregation (10/32), decreased circulating platelet aggregate ratios (5/24) and elevated plasma β-thromboglobulin levels (2/11). Several of these tests were also abnormal in diabetes mellitus thrombocytosis, leukaemia and several systemic diseases with thrombotic complications. The platelet density test using Stractan is reproducible and independent of other tests for platelet activation and is therefore potentially a useful extension of platelet function testing in patients with thrombotic disease.

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Indium-111-Labelled Human Platelets: A Method for Use in Severe Thrombocytopenia

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661183 ◽

1984 ◽

Vol 52 (03) ◽

pp. 226-229 ◽

Cited By ~ 10

Author(s):

A du P Heyns ◽

P N Badenhorst ◽

P Wessels ◽

H Pieters ◽

H F Kotzè ◽

...

Keyword(s):

Immune Thrombocytopenia ◽

Cell Layer ◽

Human Platelets ◽

Buffy Coat ◽

Severe Thrombocytopenia ◽

Red Cell ◽

Simple Method ◽

Mean Survival Time ◽

Indium 111

SummaryWe describe and evaluate a simple method for labelling autologous human platelets with Indium-111-oxine in patients with severe thrombocytopenia. Twenty patients with immune thrombocytopenia and platelet counts ranging from 5 to 119 × 109/1 were investigated. Platelets were isolated from blood by differential centrifugation, residual platelets were repeatedly washed from the red cell layer and buffy coat and labelled with In 111 in saline. A mean of 55% ± 21 of platelets were harvesteo from the blood, labelled with 49% ± 24 efficiency and 15.8 × 108 labelled platelets reinjected to the patients. Contamination of the platelets with red cells and plasma was low. The labelled platelets were viable as assessed by in vitro aggregation, recovery in the circulation and mean survival time. This method permits quantitative platelet imaging with autologous labelled platelets in patients with severe thrombocytopenia.

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Separation of Human Blood Platelets from Factor VIII (Von Willebrand’s Factor) by Combined Albumin Gradient-Gel Filtration Method

10.1055/s-0039-1680388 ◽

1977 ◽

Author(s):

S. Timmons ◽

J. Hawiger

Keyword(s):

Factor Viii ◽

Human Blood ◽

Blood Platelets ◽

Gel Filtration ◽

Human Platelets ◽

Small Samples ◽

Filtration Method ◽

Ethanesulfonic Acid ◽

Von Willebrand’S Factor

Currently used gel filtration methods were investigated in order to obtain a system which would effectively separate platelets from plasma proteins, such as Factor VIII/von Willebrand’s Factor (FVIII/vWF), in relatively small samples of human blood which are not amenable to repeated centrifugation. BioGel A150, which has a higher exclusion limit than the presently used Sepharose 2B, was found to be equally effective for preparing gel-filtered platelets. However, it did not completely separate the FVIII/vWF as judged by ristocetin-induced platelet aggregation. Therefore, a stepwise albumin gradient was employed as a preceeding step to gel filtration on BioGel A150. Platelets obtained by this modification were not responsive to ristocetin unless human plasma, as a source of FVIII/vWF, was added. Tyrode buffer, which shows variation in pH due to changes in pCO2, was replaced by the more stable zwitterionic buffer containing N-2-hydroxyethylpiperazine-N’2-ethanesulfonic acid (HEPES) pH 7.4. The need for control of CO2 was thereby obviated, reactivity of platelets in vitro was maintained, and their functional life span was extended from 1 to 2 hours. This modification of existing methods allows efficient separation of human platelets from FVIII/vWF and from other plasma macromolecules in relatively small samples of blood with preservation of the functional integrity of obtained platelet preparations.

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