Impact of Anticoagulation and Sample Processing on the Quantification of Human Blood-Derived microRNA Signatures

Blood-derived microRNA signatures have emerged as powerful biomarkers for predicting and diagnosing cardiovascular disease, cancer, and metabolic disorders. Platelets and platelet-derived microvesicles are a major source of microRNAs. We have previously shown that the inappropriate anticoagulation and storage of blood samples causes substantial platelet activation that is associated with the release of platelet-stored molecules into the plasma. However, it is currently unclear if circulating microRNA levels are affected by artificial platelet activation due to suboptimal plasma preparation. To address this issue, we used a standardized RT-qPCR test for 12 microRNAs (thrombomiR®, TAmiRNA GmbH, Vienna, Austria) that have been associated with cardiovascular and thrombotic diseases and were detected in platelets and/other hematopoietic cells. Blood was prevented from coagulating with citrate–theophylline–adenosine–dipyridamole (CTAD), sodium citrate, or ethylenediaminetetraacetic acid (EDTA) and stored for different time periods either at room temperature or at 4 °C prior to plasma preparation and the subsequent quantification of microRNAs. We found that five microRNAs (miR-191-5p, miR-320a, miR-21-5p, miR-23a-3p, and miR-451a) were significantly increased in the EDTA plasma. Moreover, we observed a time-dependent increase in plasma microRNAs that was most pronounced in the EDTA blood stored at room temperature for 24 h. Furthermore, significant correlations between microRNA levels and plasma concentrations of platelet-stored molecules pointed towards in vitro platelet activation. Therefore, we strongly recommend to (i) use CTAD as an anticoagulant, (ii) process blood samples as quickly as possible, and (iii) store blood samples at 4 °C whenever immediate plasma preparation is not feasible to generate reliable data on blood-derived microRNA signatures.

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Release of cyclic guanosine monophosphate evaluated as a diagnostic tool in cardiac diseases

Clinical Chemistry ◽

10.1093/clinchem/37.2.186 ◽

1991 ◽

Vol 37 (2) ◽

pp. 186-190 ◽

Cited By ~ 39

Author(s):

Karl-P Vorderwinkler ◽

Eilka Artner-Dworzak ◽

Gab Jakob ◽

Johanne Mair ◽

Franz Diensti ◽

...

Keyword(s):

Cyclic Guanosine Monophosphate ◽

Guanosine Monophosphate ◽

Cardiac Diseases ◽

Blood Samples ◽

Cutoff Value ◽

Intracellular Cgmp ◽

Glycerol Trinitrate ◽

Clinical Measurements ◽

And Storage

Abstract Concentrations of atrial natriuretic peptide (ANP) are increased in plasma of patients with impaired cardiac and renal function. The second messenger of ANP, cyclic guanosine monophosphate (cGMP), is released into the plasma specifically upon stimulation of cells with ANP. Although nitrates can also activate intracellular cGMP synthesis, we detected no increase in plasma cGMP concentrations after infusions of glycerol trinitrate. Because immunoreactive ANP is highly susceptible to degradation and nonspecific influences in blood samples, determinations of ANP require immediate centrifugation and storage of plasma at -20 degrees C. In contrast, we found that cGMP is stable for five days in vitro in blood samples containing EDTA. In 147 healthy blood donors, the upper cutoff value for plasma cGMP was 6.60 nmol/L, not significantly different (P greater than 0.05) from that for 222 patients with disorders other than cardiovascular and renal. In 69 patients with manifest congestive heart failure (NYHA stages II-IV), 65 had increased cGMP values. Using the above cutoff value for cGMP gave diagnostic sensitivity of 94.2% and specificity of 93.7%. Plasma cGMP may thus provide an alternative for routine clinical measurements of ANP in cardiac diseases in the absence of renal disorders.

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231 EFFECTS OF DIETARY PROPYLENE GLYCOL ON FOLLICULAR FLUID AND OVUM PICK UP IN VITRO EMBRYO PRODUCTION IN GROWTH-RESTRICTED HEIFERS WITH TWO PROFILES OF ANTI-MÜLLERIAN HORMONE

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab231 ◽

2015 ◽

Vol 27 (1) ◽

pp. 205

Author(s):

G. Gamarra ◽

C. Ponsart ◽

S. Lacaze ◽

B. Le Guienne ◽

P. Humblot ◽

...

Keyword(s):

Propylene Glycol ◽

Follicular Fluid ◽

Plasma Concentrations ◽

Oestrous Cycle ◽

Water Control ◽

Embryo Production ◽

Blood Samples ◽

Metabolic Hormones ◽

In Vitro Embryo Production

Fertility and embryo quality can be improved in cattle by using diets that induce a programmed modulation of circulating insulin concentrations. The aim of this study was to test whether the daily oral administration of propylene glycol (PG) could modify metabolite and hormone plasma and follicular fluid concentrations and improve in vitro embryo production in superovulated growth-restricted heifers (600 g day–1). Sixteen Holstein heifers were grouped according to their pre-experimental anti-Müllerian hormone (AMH) plasma concentrations: low (L = 1–80 pg mL–1; n = 7) or high (H: >150 pg mL–1; n = 9). Heifers received a single daily drench from Day 1 to Day 9 of an oestrous cycle [first cycle, 400 mL of water (control) and second cycle, 400 mL of PG]. Serial jugular blood samples were collected on Day 7 of each cycle to monitor plasma insulin, glucose, and β-hydroxybutyrate (BHB) concentrations in relation to the drench. Blood samples were also collected to measure insulin-like growth factor-1 (IGF1) and progesterone (P4) concentrations on Days 0, 2, 5, 7, and 9 of the oestrous cycle. Follicular fluid was collected on Day 9 to measure insulin and IGF1 concentrations. Ovarian ultrasonography was performed on Days 2 and 5 to count follicles between 2 and 8 mm in diameter and estimate their size. After ovum pickup (OPU) performed following superovulation on Day 5 of the oestrous cycle, oocytes were matured and fertilized in vitro, then embryos were cultured for 7 days. Propylene glycol increased plasma concentrations of insulin and glucose and reduced BHB in both groups of heifers compared with control. It also increased IGF1 concentrations on Days 5 and 7 in AMH L heifers and on Days 2, 5, and 7 in AMH H heifers, and reduced P4 concentrations on Days 5 and 9 of the oestrous cycle in all heifers. In follicular fluid, there was no difference in insulin concentrations between groups, but PG increased IGF1 concentrations in all heifers. In ovaries, PG increased the number of small follicles (2–3 mm) and total follicles on Day 2 of the cycle in all heifers, and medium follicles (4–8 mm) and total follicles on Day 5 in AMH H heifers. Propylene glycol improved the in vitro embryo development rate (total number of embryos/number of fertilized oocytes) in all heifers (AMH L: control, 37.9% v. PG, 50.0%; P < 0.05; AMH H: control, 36.4% v. PG, 48.3%; P < 0.05). In AMH H, the number of grade 1 blastocysts was increased by PG (control, 5.2 ± 1.0 v. PG, 8.9 ± 1.0; P < 0.01), whereas there was no difference between treatments in AMH L heifers (control, 1.9 ± 1.1 v. PG, 3.2 ± 1.1; P > 0.05). These results indicate that short-term oral PG supplementation affects the concentrations of metabolites and metabolic hormones in blood and IGF1 concentrations in follicular fluid. PG administration is effective in improving in vitro embryo production more markedly in heifers with high AMH compared with low AMH endocrine levels.

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Stability of Parathyroid Hormone-Related Protein and Parathyroid Hormone at Room Temperature

Annals of Clinical Biochemistry International Journal of Laboratory Medicine ◽

10.1177/000456329403100513 ◽

1994 ◽

Vol 31 (5) ◽

pp. 497-500 ◽

Cited By ~ 19

Author(s):

G E Levin ◽

J A Nisbet

Keyword(s):

Parathyroid Hormone ◽

Protease Inhibitor ◽

Room Temperature ◽

Related Protein ◽

Blood Samples ◽

Parathyroid Hormone Related Protein ◽

Edta Plasma ◽

The Stability ◽

Serum Pth ◽

Zero Time

The stability of plasma parathyroid hormone-related protein (PTHrP) as measured by the Nichols Institute assay at room temperature was assessed over a period of 72 h in blood samples collected in protease inhibitor tubes and EDTA tubes at 0, 6, 24, 48 and 72 h from 10 patients with hypercalcaemia of malignancy. Mean plasma PTHrP concentrations in blood samples collected in protease inhibitor tubes remained stable for up to 48 h but had decreased by 10% at 72 h. The mean EDTA plasma PTHrP at zero time was 67% of the protease inhibitor tube value and this had fallen to 39% at 72 h. The stability of parathyroid hormone (PTH) in separated blood samples was also assessed by collection into heparin and plain tubes as well as EDTA and protease inhibitor tubes. Serum PTH concentrations progressively declined throughout the 72 h study period although the zero time values were significantly higher than corresponding plasma PTH concentrations. Plasma PTH concentrations appeared to be stable when blood was collected in heparin, EDTA and protease inhibitor tubes during the 72 h period, except in one subject with markedly elevated plasma amylase activity.

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Differences of Phenylalanine Concentrations in Dried Blood Spots and in Plasma: Erythrocytes as a Neglected Component for This Observation

Metabolites ◽

10.3390/metabo11100680 ◽

2021 ◽

Vol 11 (10) ◽

pp. 680

Author(s):

Dorothea Haas ◽

Jana Hauke ◽

Kathrin V. Schwarz ◽

Lucia Consalvi ◽

Friedrich K. Trefz ◽

...

Keyword(s):

Mass Spectrometry ◽

Venous Blood ◽

Plasma Concentrations ◽

Dried Blood Spots ◽

Ion Exchange Chromatography ◽

Exchange Chromatography ◽

Tandem Mass ◽

Blood Samples ◽

Blood Spots ◽

Edta Plasma

Monitoring phenylalanine (Phe) concentrations is critical for the management of phenylketonuria (PKU). This can be done in dried blood spots (DBS) or in EDTA plasma derived from capillary or venous blood. Different techniques are used to measure Phe, the most common being flow-injection analysis tandem mass spectrometry (FIA-MS-MS) and ion exchange chromatography (IEC). Significant differences have been reported between Phe concentrations in various sample types measured by different techniques, the cause of which is not yet understood. We measured Phe concentrations in 240 venous blood samples from 199 patients with hyperphenylalaninemia in dried blood spots, EDTA plasma and erythrocytes by FIA-MS-MS and IEC. Phe concentrations were significantly lower in erythrocytes than in plasma leading to about 19% lower Phe DBS concentrations compared with plasma independent from the method used for quantification. As most therapy recommendations for PKU patients are based on plasma concentrations reliable conversion of DBS into plasma concentrations is necessary. Variances of Phe concentrations in plasma and DBS are not linear but increases with higher concentrations indicating heteroscedasticity. We therefore suggest the slope of the 75th percentile from quantile regression as a correction factor.

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Preanalytical stability of [-2]proPSA in whole blood stored at room temperature before separation of serum and plasma: implications to Phi determination

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2018-0596 ◽

2019 ◽

Vol 57 (4) ◽

pp. 521-531 ◽

Cited By ~ 2

Author(s):

Ruggero Dittadi ◽

Aline S.C. Fabricio ◽

Giulia Rainato ◽

Edoardo Peroni ◽

Fulvio Di Tonno ◽

...

Keyword(s):

Whole Blood ◽

Room Temperature ◽

Specific Antigen ◽

Free Psa ◽

Glass Tubes ◽

Edta Plasma ◽

Total Psa ◽

Clot Activator ◽

Over Time

Abstract Background [-2]proPSA seems to outperform free/total prostate-specific antigen (PSA) ratio in prostate cancer diagnosis. However, [-2]proPSA stability remains an underestimated issue. We examined [-2]proPSA stability over time in whole blood before separation of serum and plasma and its implications for prostate health index (Phi) determination. Total PSA (tPSA) and free PSA (fPSA) stabilities were also assessed. Methods Blood was drawn from 26 patients and separated in two tubes for plasma (K2EDTA and K2EDTA plus protease inhibitors – P100) and one for serum (clot activator plus gel separator). Tubes were stored at room temperature before centrifugation 1, 3 and 5 h for serum and EDTA plasma or 1 and 5 h for P100 plasma. To investigate the influence of gel separator on markers’ stability, blood was collected from 10 patients in three types of tubes to obtain serum: tubes with clot activator plus gel separator, with silica particles or glass tubes. Biomarkers were assayed with chemiluminescent immunoassays. Results [-2]proPSA and Phi levels significantly and progressively increased over time in serum (+4.81% and +8.2% at 3 h; +12.03% and +14.91% at 5 h, respectively, vs. 1 h; p<0.001). Conversely, [-2]proPSA levels did not change in plasma (EDTA or P100). tPSA levels did not change over time in serum or plasma, whereas fPSA decreased in serum. All markers were higher in plasma than in serum at any time point. This difference did not seem to be attributable to the use of gel for serum preparation. Conclusions EDTA prevented spurious in vitro modifications in PSA-related isoforms, confirming that a stabilized blood sample is a prerequisite for [-2]proPSA measurement and Phi determination.

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Assessment of the stability of N-terminal pro-brain natriuretic peptide in vitro: implications for assessment of left ventricular dysfunction

Clinical Science ◽

10.1042/cs0970255 ◽

1999 ◽

Vol 97 (3) ◽

pp. 255-258 ◽

Cited By ~ 24

Author(s):

P. F. DOWNIE ◽

S. TALWAR ◽

I. B. SQUIRE ◽

J. E. DAVIES ◽

D. B. BARNETT ◽

...

Keyword(s):

Clinical Practice ◽

Brain Natriuretic Peptide ◽

Natriuretic Peptide ◽

Left Ventricular Dysfunction ◽

Room Temperature ◽

Ventricular Dysfunction ◽

Plasma Concentrations ◽

Left Ventricular ◽

Blood Samples ◽

The Stability

Plasma concentrations of N-terminal pro-brain natriuretic peptide (NT-proBNP) are raised in patients with left ventricular dysfunction. Measurement of this peptide has a potential diagnostic role in the identification and assessment of patients with heart failure. The stability of this peptide over time periods and conditions pertaining to routine clinical practice has not been reported previously. Blood samples were obtained from 15 subjects. One aliquot was processed immediately, and the remaining portions of the blood samples were stored for 24 h or 48 h at room temperature or on ice prior to processing. Plasma concentrations of NT-proBNP were measured with a novel immunoluminometric assay developed within our laboratory. Mean plasma concentrations of NT-proBNP were not significantly different whether blood samples were centrifuged immediately and stored at -70 °C or kept at room temperature or on ice for 24 h or 48 h. The mean percentage differences from baseline (reference standard) were +5.2% (95% confidence interval +18.2 to -7.8%) and +0.8% (+15.2 to -13.7%) after storage for 24 h at room temperature or on ice respectively, and +8.9% (+24.2 to -6.5%) and +3.2% (+15.1 to -0.9%) for storage for 48 h at room temperature or on ice respectively. Pearson correlation coefficients for baseline NT-proBNP concentrations compared with levels at 48 h at room temperature or on ice were r = 0.89 and r = 0.83 respectively (both P < 0.0001). Thus NT-proBNP extracted from plasma samples treated with EDTA and aprotinin is stable under conditions relevant to clinical practice.

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Possible Mechanism for in Vitro Complement Activation in Blood and Plasma Samples: Futhan/EDTA Controls in Vitro Complement Activation

Clinical Chemistry ◽

10.1093/clinchem/45.8.1190 ◽

1999 ◽

Vol 45 (8) ◽

pp. 1190-1199 ◽

Cited By ~ 52

Author(s):

Philippe H Pfeifer ◽

Marleen S Kawahara ◽

Tony E Hugli

Keyword(s):

Liver Transplant ◽

Complement Activation ◽

Room Temperature ◽

Plasma Samples ◽

Accurate Analysis ◽

Transplant Patients ◽

Edta Plasma ◽

The Mean

Abstract Background: Ongoing in vitro complement (C) activation in citrate or EDTA plasma has prevented an accurate analysis of C-activation products generated in vivo. The aim of this study was to characterize handling and storage conditions required to prevent in vitro C activation in blood and plasma samples collected with Futhan/EDTA. Methods: BiotrakTM RIAs were used to quantitatively measure C3a and C4a in blood and/or plasma samples from healthy individuals (controls) and from liver transplant patients. Blood samples were routinely drawn into either EDTA (1 g/L) tubes or into tubes containing both EDTA (1 g/L) and Futhan (0.1 g/L) and immediately centrifuged at 2000g for 15 min at 4 °C. Results: In controls, C4a, but not C3a, in fresh samples (time 0) was higher in EDTA plasma than in Futhan/EDTA plasma (n = 20; P = 0.002). Futhan/EDTA prevented C3a and C4a generation in blood and plasma samples held at room temperature (22–23 °C) for 1 h and in plasma held for 24 h at 4 °C or −70 °C. The mean C3a concentration (1.76 mg/L; n = 19) at time 0 in EDTA plasma samples from liver transplant patients was significantly higher than for controls (0.34 mg/L; n = 11). In these patients, the mean C3a in EDTA samples increased to 13.8 mg/L after 60 min at room temperature, but there was no change in the C3a concentration of an EDTA plasma from a control. In the patients, C3a concentrations were lower in Futhan/EDTA plasma than in EDTA at time 0 and after 60 min at room temperature (1.40 and 2.02 mg/L, respectively). The mean patient C4a was 4.02 mg/L in EDTA plasma at time 0 vs 0.24 mg/L for controls; it increased to 16.9 mg/L after 60 min at room temperature compared with 0.76 mg/L for controls. The mean patient C4a was 0.83 mg/L in Futhan/EDTA plasma at time 0 vs 0.1 mg/L for controls. Neither patient nor control C4a concentrations increased vs time in Futhan/EDTA. Conclusion: The combination of Futhan (0.1 g/L) and EDTA (1 g/L) eliminates in vitro C activation.

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THE SIGNIFICANCE OF VON WILLEBRAND FACTOR ANTIGEN (vWFAg) AS AN ENDOTHELIAL MARKER DURING HAEMODIALYSIS

10.1055/s-0038-1643345 ◽

1987 ◽

Author(s):

P B Rylance ◽

M P Gordge ◽

N J Dood ◽

M J Weston

Keyword(s):

Molecular Weight ◽

Platelet Activation ◽

Plasma Concentrations ◽

Regional Citrate Anticoagulation ◽

Low Molecular Weight ◽

Von Willebrand ◽

Willebrand Factor ◽

Factor Antigen ◽

Endothelial Marker

It has been suggested that platelet activation during haemodialysis (HD) is associated with damage to the endothelium which might contribute to increased atheroma seen in HD patients. We have therefore measured vWFAg (by radial immunodiffusion) as an endothelial marker during HD and B-thromboglobulin (BTG) (by RIA) as a marker of platelet activation with various anticoagulant regimes: unfractionated heparin (UFH) (Leo), low molecular weight heparin (LMWH) (Choay CY 216), regional citrate anticoagulation, prostacyclin (PGI2) (5ng/kg/min) and the combination of PGI2 + UFH.Platelet activation occurred (as shown by a progressive rise of BTG) during HD with UFH, LMWH and citrate, but not with PGI2 or PGI2 + UFH. Pre-dialysis vWFAg levels were elevated in HD patients (2.37 ± 1.16 (SD) U/ml) compared with healthy controls (1.15 ±0.34 U/ml, p<0.001). Plasma concentrations of vWFAg increased during HD with UFH (26 ± 13* rise after 120 mins, p<0.001), but not during HD with LMWH, citrate, PGI2 or PGI2 + UFH. UFH infusion without HD did not result in vWFAg rise nor did UFH or IMWH interfere in vitro with the vWFAg assay. Thus PGI2 prevented both platelet activation and release of vWFAg. With IMWH and citrate there was no increase of vWFAg despite evidence of platelet activation.Elevated pre - dialysis vWFAg concentrations in HD patients may reflect chronic endothelial injury or alternatively may be due to impaired clearance of vWFAg in uraemia. The results obtained during HD suggest that vWFAg release occurred only in the presence of both platelet activation and high molecular weight heparin chains. Whether the release of vWFAg reflects disruption and damage to the endothelium or stimulation and displacement of vWFAg remains uncertain.

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Prevention of in Vitro Lipolysis by Tetrahydrolipstatin

Clinical Chemistry ◽

10.1093/clinchem/46.7.950 ◽

2000 ◽

Vol 46 (7) ◽

pp. 950-954 ◽

Cited By ~ 48

Author(s):

Michael Krebs ◽

Harald Stingl ◽

Peter Nowotny ◽

Daniel Weghuber ◽

Martin Bischof ◽

...

Keyword(s):

Room Temperature ◽

Storage Temperature ◽

Lipolytic Activity ◽

Accurate Determination ◽

Plasma Concentrations ◽

Metabolic Effects ◽

In Vitro Lipolysis ◽

Plasma Ffa ◽

Plasma Ffas

Abstract Background: Metabolic effects of free fatty acids (FFAs) frequently are tested using combined infusion of triglycerides and heparin, which stimulates lipolysis in vivo. Ongoing in vitro lipolysis, however, probably produces falsely high plasma FFA concentrations under these conditions. Therefore, this study aims to assess the efficacy of tetrahydrolipstatin (THL) in inhibiting plasma lipolytic activity and to improve plasma FFA determination. Methods: Plasma concentrations of FFAs and glycerol were measured in five healthy subjects in the presence and absence of THL. Blood was drawn at baseline, during infusion of a triglyceride emulsion (1.5 mL/min), and during infusion of triglycerides plus heparin (0.2 IU · kg−1 · min−1). In addition, the effects of storage temperature of the samples were analyzed. Results: In samples frozen immediately after collection, plasma FFAs were 28% lower in the presence of THL than in its absence (P = 0.008). When THL-free plasma was incubated for 3 h on ice or at room temperature, plasma FFAs were 22% (P = 0.02) and 91% (P = 0.0004) higher, respectively, than in samples frozen immediately. The addition of THL blunted temperature-dependent in vitro lipolysis by 88% (P <0.01) and 89% (P <0.001) after incubation on ice and at room temperature, respectively. Changes in plasma glycerol concentrations exhibited similar behavior. Conclusions: THL, which is safe and easy to handle, is a potent inhibitor of in vitro lipolysis and could, therefore, be added to blood samples drawn during triglyceride/heparin infusions to allow more accurate determination of plasma FFA concentrations.

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P-Selectin- and CD63-Exposing Platelet Microparticles Reflect Platelet Activation in Peripheral Arterial Disease and Myocardial Infarction

Clinical Chemistry ◽

10.1373/clinchem.2005.057414 ◽

2006 ◽

Vol 52 (4) ◽

pp. 657-664 ◽

Cited By ~ 128

Author(s):

P Marc van der Zee ◽

Éva Biró ◽

Yung Ko ◽

Robbert J de Winter ◽

C Erik Hack ◽

...

Keyword(s):

Myocardial Infarction ◽

Peripheral Arterial Disease ◽

Platelet Activation ◽

Plasma Concentrations ◽

Arterial Disease ◽

Thrombin Receptor ◽

Ionophore A23187 ◽

St Elevation ◽

Peripheral Arterial

Abstract Background: Platelet-derived microparticles (PMPs) are generally considered a marker of platelet activation in cardiovascular disease. We studied the extent to which PMP subpopulations parallel platelet activation in vitro and in vivo. Methods: Using flow cytometry, we analyzed PMP subpopulations from resting and activated platelets in vitro (n = 6) as well as from plasma samples of patients with stable angina, peripheral arterial disease, or myocardial infarction [non-ST-elevation (NSTEMI) and ST-elevation (STEMI)] and from older, age- and sex-matched and young healthy individuals [n = 10 for all groups except NSTEMI (n = 11)]. Coagulation markers prothrombin fragment F1 + 2 and thrombin-antithrombin complexes were determined by ELISA. The PMP-associated fraction of soluble (s)P-selectin was estimated by ELISA. Results: In vitro, stimulation of platelets with thrombin receptor–activating peptide (15 μmol/L) or the calcium ionophore A23187 (2.5 μmol/L) increased fractions of both platelets and PMPs exposing P-selectin or CD63 (P <0.001 for all). Whereas the number of PMPs released by A23187-stimulated platelets increased significantly (P <0.001), the number of PMPs released from thrombin receptor-activating peptide–stimulated platelets remained constant (P >0.05). Ex vivo, numbers of circulating PMPs were comparable in all groups. Compared with young persons, P-selectin–exposing PMPs were increased in older persons (P = 0.02) and were further increased in patients with NSTEMI (P = 0.007) and STEMI (P = 0.045). CD63-exposing PMPs were increased in patients with peripheral arterial disease (P = 0.041), NSTEMI (P = 0.001), and STEMI (P = 0.049). Subpopulations exposing P-selectin or CD63 correlated with each other (r = 0.581; P <0.001), but neither correlated with the plasma concentrations of F1 + 2 or thrombin–antithrombin complexes. The PMP-associated fraction of sP-selectin constituted only 2.2 (4.7)% [mean (SD)] of total sP-selectin. Conclusions: PMP subpopulations reflect platelet activation status better than the total number of PMPs. Increased concentrations of circulating PMP subpopulations are found in aging, and further increases are encountered in peripheral arterial disease and myocardial infarction.

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